Caterpillar Labial Saliva Alters Tomato Plant GeneExpression

Richard O. Musser & Sue M. Hum-Musser & Henry K. Lee &

Brittany L. DesRochers & Spencer A. Williams & Heiko Vogel

Received: 15 June 2012 /Revised: 7 September 2012 /Accepted: 25 September 2012# Springer Science+Business Media New York 2012

Abstract We examined the effects of Helicoverpa zea cater-pillar labial saliva on tomato plant gene expression. Caterpil-lars with labial salivary glands (mock-ablated) and without(ablated) were fed on tomato plants for 24 hr; then, the leafmRNAwas analyzedwith tomatomicroarrays. Analysis of thetranscript profiles revealed 384 expressed sequence tags(ESTs) that were significantly altered due to herbivory com-pared to the non-wounded plants. The majority of the ESTswere quantitatively altered more so by mock-ablated caterpil-lars with labial salivary glands than ablated caterpillars. Par-ticularly notable, ESTs encoding acid phosphatase, arginase,acidic endochitinase, dehydrin, polyphenol oxidase, proteaseinhibitors, and threonine deaminase were more highly stimu-lated by mock-ablated caterpillars than ablated caterpillars. Inaddition, tomato leaves were mechanically wounded withscissors and painted with labial salivary gland extract, auto-claved salivary gland extract, or water, and compared to non-wounded tomato plants. After 4 hr, these leaves were collectedand a tomato microarray analysis of the mRNA revealedcorrelation of the gene expression of these leaves altered bymechanical wounding and painted with salivary gland extractto the gene expression of leaves fed on by mock-ablatedcaterpillars. We show that caterpillar labial saliva is an

important component of herbivory that can alter plant geneexpression.

Keywords Caterpillar saliva .Herbivory .Gene expression .

Glucose oxidase . Plant defense

Introduction

Increasing evidence suggests that plants, to some degree, candifferentiate between tissue damage caused by environmentalfactors (wind, hail) and actual insect herbivory that wouldresult in a more continuous loss of tissues (Howe and Jander,2008). The herbivore’s unique style and tempo of woundingalso can function as a reliable signal of herbivory. Mithöfer etal. (2005) utilized a computer-controlled tool, “MecWorm,”that realistically mimicked caterpillar wounding and stimulat-ed Lima bean plant volatiles similar to the actual caterpillar.However, further MecWorm experiments revealed that vola-tile expression is altered by additional herbivory factors, suchas insect oral secretions, beyond physical plant tissue damage(Maffei et al., 2007a,b).

Insect oral secretions contain many different factors thatpotentially affect a wide range of plant physiology, and canfunction as elicitors that stimulate the plant’s anti-herbivoredefenses (Mattiacci et al., 1995; Alborn et al., 1997, 2000;Reymond et al., 2000, 2004; Hermsmeier et al., 2001; Schittkoet al., 2001; Lou and Baldwin, 2003; De Vos et al., 2005, 2006;Ralph et al., 2006a,b; Schmelz et al., 2006, 2009; Lawrence etal., 2008). Herbivore elicitors can include many compoundssuch as enzymes (β-glycosidase, glucose oxidase (GOX),ribonuclease), small peptides called inceptins, fatty acid-amino acid conjugates such as volicitin, and sulfated !-hy-droxy fatty acids called caeliferins (Musser et al., 2002a, b,2005a, b; Howe and Jander, 2008; Mithöfer and Boland,2008). Musser et al. (2002a, 2005a, b, 2006) first characterized

Electronic supplementary material The online version of this article(doi:10.1007/s10886-012-0198-3) contains supplementary material,which is available to authorized users.

R. O. Musser (*) : S. M. Hum-Musser :H. K. Lee :B. L. DesRochers : S. A. WilliamsDepartment of Biological Sciences, Western Illinois University,Macomb, IL 61455, USAe-mail: RO-Musser@wiu.edu

H. VogelDepartment of Entomology,Max Planck Institute for Chemical Ecology,Beutenberg Campus,Jena 07745, Germany

J Chem EcolDOI 10.1007/s10886-012-0198-3

GOX in Helicoverpa zea labial saliva as a “suppressor” ofinducible nicotine, an anti-herbivore defense of Nicotianatabacum (cultivated tobacco). The labial saliva of many cater-pillar species contains GOX, which catalyzes the oxidation ofD-glucose, producing hydrogen peroxide (H2O2) and D-gluconic acid (Felton and Eischenseer, 1999; Eischenseeret al., 1999, 2010).

Genomic tools have been utilized to investigate herbivore-associated molecular patterns (HAMPs) (Mithöfer andBoland, 2008). One of the first plant-insect microarray studiescompared the gene expression of Arabidopsis following Pie-ris rapae caterpillar herbivory to the gene expression of me-chanically wounded leaves (Reymond et al., 2000; Korth,2003). It was determined that compared to wounding alone,caterpillar herbivory differentially affected the expression of anumber of genes, in particular, dehydration-inducible genesand the stimulation ofHel, a latex gene, which appeared to beherbivory-specific (Reymond et al., 2000).

Other studies have demonstrated the induction of specificplant gene expression by herbivore oral secretions. Volicitin, acompound of Spodoptera exigua (beet armyworm) regurgi-tant, applied to wounded maize leaves, caused an up-regulation of gene expression related to the synthesis of vol-atile compounds (Lawrence and Novak, 2004). Spodopteraexigua salivary factors altered Medicago truncatula mRNAtranscripts encoding terpene synthases (Gomez et al., 2005;Bede et al., 2006). Several microarray studies of Nicotianaattenuata (wild tobacco) suggest that fatty acid-amino acidconjugates of Manduca sexta caterpillar regurgitant are re-sponsible for stimulating jasmonate-related plant defensegenes (Skibbe et al., 2008). Spodoptera exigua fatty acid-amino acid conjugates also stimulated jasmonate-related plantdefenses; however, these responses were altered by the pres-ence of GOX, which tended to stimulate salicylate plantresponses and attenuate the expression of jasmonic acid (JA)defense genes (Diezel et al., 2009). This also was shown bythe response of Arabidopsis thaliana to S. exigua herbivorywhere the presence of GOX in the saliva appeared to stimulatesalicylate responses (Weech et al., 2008) but was not seen intomato plants (Tian et al., 2012). Ultimately, it is to the plant’sadvantage to determine the attacker and formulate a specificdefense response, while it is advantageous for the herbivore tominimize detection (Glazebrook, 2005; Koornneef andPieterse, 2008; Diezel et al., 2009; Leon-Reyes et al., 2009).

Most of the studies on HAMPs use regurgitant, whichgenerally contains a mixed variety of factors including man-dibular and labial saliva, consumed and partially digestedplant material, gut enzymes, other gut contents, and bacteria.Other studies that have focused on only caterpillar labial salivademonstrate that saliva can alter plant defenses (Musser et al.,2002a, b, 2005a, 2006; Bede et al., 2006; Delphia et al., 2006;Darwish et al., 2008; Weech et al., 2008). In this study, weused global gene expression analysis to examine the effect of

H. zea caterpillar labial saliva on tomato plant responses.Surgical removal of caterpillar labial salivary glands allowedus to specifically control and address how labial saliva affectsplant responses, separate from the physical damage due tocaterpillar feeding and not just the addition of oral secretions.We hypothesized that caterpillar labial saliva would altertomato plant gene expression differently than herbivory with-out salivary secretions. Helicoverpa zea caterpillars with(mock-ablated) and without (ablated) labial salivary glandswere fed on tomato plants for 24 hr ad libitum. Caterpillarsalso were placed in cages on tomato leaflets (for 12 and 24 hr)to more carefully control the extent of damage of the tomatoleaf. In addition, labial salivary gland extract was applied tomechanically wounded tomato leaves and compared to leaveswounded and treated with water or autoclaved salivary glandextract. This study examined global tomato plant transcrip-tome alterations due to caterpillar labial saliva.

Methods and Materials

Tomato Plants Solanum lycopersicum (Big Boy) tomatoseeds were planted in 500 ml plastic pots in SunshineSB300 potting mix (SunGro Horticulture Distribution, Inc,Bellevue, WA, USA). Plants were grown in 15:9 L:D cyclesat 33 °C, watered as needed, and fertilized (N:P:K015:30:15, American Plant Food CO., Creve Coeur, MO,USA) weekly. Healthy, undamaged, 5-wk-old plants at 12inches in height, with three fully expanded leaves, wereused in the experiments.

Caterpillar Rearing, Surgery, and Herbivory on TomatoPlants Helicoverpa zea eggs were initially acquired fromthe NC State University insectary for the first 24 hr herbivorymicroarray experiments, and were subsequently obtainedfrom Dr. Patrick Dowd (USDA-ARS, Peoria, IL, USA) forthe follow-up 12-h and 24-h herbivory experiments analyzedwith real-time quantitative polymerase chain reaction (qPCR).The caterpillar neonates were placed into 2-ounce medicinecups filled with corn-based caterpillar diet (Chippendale,1970; Broadway and Duffey, 1986). Neonates were rearedin a 15-h photoperiod at 33 °C until the 2nd day of the 6thinstar when the labial salivary glands were removed by sur-gery (Musser et al., 2005a, 2006). To determine the effect oflabial saliva on tomato plants in situ, labial salivary glandswere removed from 50 caterpillars (ablated) (Musser et al.,2006). A mock-ablated surgery was performed on another setof 50 caterpillars (mock-ablated) where the glands were leftintact after the same incision was made. After 5 hr of recovery,5 caterpillars of each type were placed onto each tomato plantto feed ad libitum for a total of 5 plants per respective treat-ment in the plant growth chamber. The larvae were checkedperiodically; careful selection of caterpillars that were good

J Chem Ecol

feeders and replacement of caterpillars that were unsatisfacto-ry feeders was done to ensure similar herbivory rates. A thirdset of 5 plants were left as non-wounded controls. Each plantwas a separate replicate. After 24 hr, only leaflets with obviouswounding and similar amounts of damage (~ half a leaflet/leaflet) were removed, pooled for each individual plant, flashfrozen, and stored at !80 °C until processed. The experimentwas replicated a 2nd time.

Follow-up Tomato Plant Caterpillar Herbivory Experi-ment For more stringent control of herbivory betweenmock-ablated and ablated caterpillars, the experiment wasrepeated as described above; however, these caterpillars wereplaced in clip cages to restrict the amount of herbivory to halfa leaflet each on 3 different leaves per tomato plant. Damagedleaflets from each plant were harvested at 12 hr and 24 hr post-herbivory, pooled, and frozen. There was a minimum of fourbiological replicates for each time point. Gene expression wasmeasured with qPCR on all the biological replicates.

Labial Salivary Gland Extract Preparation Labial salivaryglands were harvested from 2nd-day-old 6th instar caterpil-lars as described by Musser et al. (2005a, b, 2006). Thesalivary glands (30–50 pairs) were combined into a micro-centrifuge tube and stored at !80 °C until used. Labialsalivary glands were thawed, homogenized, and centrifugedat 10,000x g for 5 min. Protein concentration in the homog-enate was determined with a Bradford assay that was scaleddown in volume (100 ul) and read with a microplate reader(Bradford, 1976; Dynex MRX II, Dynex Technologies, Inc.,Chantilly, VA, USA). Salivary gland extracts were diluted to0.5 mg/ml total protein concentration.

Effect of Caterpillar Labial Salivary Gland Extracts onTomato Experiment Five leaflets on 3 of the upper mostfully expanded leaves were wounded with fabric scissorswith jagged v-shaped edges to simulate caterpillar feeding.Approximately half of each leaflet was removed. Immedi-ately after wounding, the leaf’s wounded edge was paintedwith 20ul of 0.5 mg/ml protein concentration of one of thefollowing: labial salivary gland extract (sal), autoclavedsalivary gland extract (auto), GOX (1 mg/mL; Sigma Chem-ical Co., St. Louis, MO, USA), or distilled water (wat). Anon-wounded (nw) control was used to measure the relativechanges in the plants from wounding. Based on Peiffer andFelton (2005) 0.5ug of GOX is applied per hour by H. zeacaterpillars, so roughly the equivalent of 10 to 20 hr ofsalivary secretions were applied to each wound. After 4 hr,the leaflets from each plant were pooled, flash frozen inliquid nitrogen, and stored at !80 °C until used for geneexpression analysis. For microarrays, only nw-, sal-, auto-,and wat-treated leaflets were used with a minimum of 4biological replications, and qPCR was conducted to confirm

gene expression patterns for genes of interest for all thetreatments described.

cDNA Tomato Microarray Protocol The RNA preparationand microarray handling procedures were similar to proto-cols described in Rodriguez-Saona et al. (2010), except forthe differences detailed below. The TOM1 cDNA micro-array and corresponding gene annotations from the BoyceThompson Institute (Ithaca, NY, USA) contain 12,899 ESTclones and an estimated 8,500 unique genes (Alba et al.,2004).

Extraction of Total RNA and Messenger RNA Total RNAwas extracted from 5 g of frozen leaf material for each samplewith TRIzol® (Invitrogen Corporation by Life Technologies,Carlsbad, CA, USA) following the manufacturer’s protocol.The reagent volumes for the remainder of the procedure werescaled up accordingly. The total RNA pellet was air dried, andthen resuspended in 400 μl of RNase-free water. The totalRNA was quantified with a Nanodrop 2000 (Thermo Scien-tific, Wilmington, DE, USA) and stored at !80 °C. MessengerRNAwas purified from 300 μg of total RNA using the Oligo(dT) Dynalbeads (Invitrogen) according to the manufacturer’sprotocol. The mRNAwas eluted in 80 μl of 10 mMTris–HCl,pH 7.5, quantified, and then stored at !80 °C.

Direct Labeling of RNA and Hybridization of the LabeledcDNA onto Microarray Slides Complementary DNA(cDNA) was reverse-transcribed from 3 μg mRNA withSuperscript II (Invitrogen), and labeled with Cy3 or Cy5dyes according to the manufacturer’s protocol (AmershamBiosciences, Piscataway, NJ). Hybridization was performedusing 40 μl of the Cy3- and Cy5-labeled targets on theTOM 1 tomato microarray chip for 10 hr at 60 °C. Thetomato microarrays were washed, dried, and then scannedimmediately.

Layout for the Microarray Chips We used a replicated loopdesign for microarray hybridizations, where each samplewas hybridized with every other sample, and included dyereversal hybridizations for the samples. (Townsend, 2003).The dye swaps were performed to correct for dye-biasedcDNA labeling. Five microarrays were successfully com-pleted for the caterpillar herbivory experiment: tomatoleaves damaged and fed on by caterpillars with labial sali-vary glands (mock-ablated) (4 biological replications), with-out (ablated) (3 biological replications), or the non-woundedcontrol (nw) (3 biological replications). Eleven microarraychips were successfully completed for the four tomato planttreatments used in the simulated caterpillar wounding ex-periment: wounded (scissors) and treated with labial sali-vary gland extract (sal) (9 biological replications), woundedand treated with autoclaved labial salivary gland extract

J Chem Ecol

(auto) (6 biological replications), wounded and treated withdistilled water (wat) (3 biological replications), or non-wounded control (nw) (4 biological replications). Two treat-ments were placed onto each microarray (Cy3 versus Cy5).

Microarray Scanning and Analysis of Tomato MicroarrayData The microarrays were scanned with a Gene Pix 4100A microarray scanner (Axon Molecular Devices, UnionCity, CA, USA) at Western Illinois University using theGenePix Pro 6.0 software (Axon Molecular Devices). Themicroarrays were scanned at the appropriate wavelengths toobtain each spot’s signal intensity, which indicated levels ofcDNA that had hybridized to each DNA spot on the chip.The photomultiplier detectors were manually set to equalout the overall intensities of the Cy3 and Cy5 scans.

Statistical Analysis of Microarray Data We used the Gene-sifter™ software (Geospiza, Inc., Seattle, WA, USA) toanalyze the overall gene expression. Data from each micro-array were log2 scaled and normalized with the genomicmedian intensity. A one-way ANOVA for Multiple Groupswas performed with a Benjamini and Hochberg False Dis-covery Rate (FDR) statistical correction that controls fam-ilywise error rate in multiple hypotheses testing that occursin microarray studies (Benjamini and Hochberg, 1995). As apost-hoc test to the ANOVA, we used the conservativeTukey’s Honestly Significant Differences (HSD) test forgenes of interest at P-value of <0.05. As a supplementaryanalysis, we determined the q-value for each treatment’sgene expression, which allowed for the adjustment for thefalse discovery rate (Storey and Tibshirani, 2003). Thesestatistics were additionally verified with the SignificanceAnalysis of Microarrays (SAM) statistical software (http://www-stat.stanford.edu/~tibs/SAM/) and with the R statisti-cal package (http://www.r-project.org/).

Real-time Quantitative Polymerase Chain Reaction to Con-firm Microarray Results Total RNA was processed forqPCR as described in Rodriguez-Saona et al. (2010). TheVerso cDNA Synthesis Kit (Thermo Fisher Scientific,Waltham, MA, USA) was used to synthesize cDNA from1 μg of total RNAwith oligo-d(T) primers for 1 hr at 42 °Caccording to manufacturer’s instructions. The qPCR wasdone with the Fast SYBR® Green Master Mix (AppliedBiosystems by Life Technologies, Carlsbad, CA, USA) inoptical 96-well plates in the StepOne Plus Real-Time PCRSystem instrument (Applied Biosystems). The qPCR wasdone for several plant defense genes (primer sequences andgene abbreviations are listed in Table 1). The actin geneamplicon was used as the endogenous control, and dataanalysis was completed as described in Rodriguez-Saonaet al. (2010). A minimum of four biological replicates wereconducted for all the assays.

Results

Overall Genomic Expression of Caterpillar HerbivoryExperiments Using the Benjamin-Hochberg False DiscoveryRate (FDR adjusted P<0.05) a multiple test correction used inmicroarray experiments, there were 384 expressed sequencetags (ESTs) that were significantly altered 24 hr post herbivorycompared to the non-wounded control (SupplementalFile 1). Of these 384 ESTs, 155 were unknown, 85 werephotosynthesis-related, and 60 were divided into plantdefense-, plant pathogen-, and abiotic-related genes based ona literature review of the EST annotations. As expected, her-bivory generally up-regulated plant defense, abiotic response,and pathogenesis-related genes, and generally down-regulatedESTs for photosynthesis and metabolism (Tables 2 and 3). Inmost cases, the differences in the stimulation or suppression oftomato plant genes were generally greater from herbivory bycaterpillars with labial salivary glands in comparison to cater-pillars without labial salivary glands. The majority of quantita-tive differences in plant gene expression between mock-ablatedand ablated caterpillars were related to anti-nutritive defensessuch as acid phosphatase, arginase, polyphenol oxidase F (PPOF), protease inhibitors, and threonine deaminase (Table 2).These genes also were significantly more stimulated by cater-pillars that could secrete labial saliva vs. the ablated caterpillars.In addition, the expression of several tomato plant genes werequalitatively stimulated at higher levels of expression bymock-ablated caterpillars compared to ablated caterpillars, such asacidic 26 kDa endochitinase precursor, dehydrin, pathogenesis-related leaf protein 6, beta-carbonic anhydrase, and anthranilatephosphoribosyltransferase (Table 2).

As an additional comparative analysis that may be help-ful to some researchers for future investigations, we haveincluded genes with different expression analyzed with aless conservative method than the Benjamin-HochbergFDR, albeit still using q-values that were determined withthe Storey Method (Supplemental File 3) which is anothermanner to control for the FDR in microarray studies (Storeyand Tibshirani, 2003). We found substantial overlap ofgenes with the previous analysis, however, the expressionof more genes was altered using a FDR of 4.81 %, where734 ESTs were significantly different.

Hierarchal Cluster of Caterpillar Herbivory Experiments Ahierarchal cluster using the Manhattan Method (GeneSifterTM)for the 384 ESTs [(P<0.05; FDR corrected ANOVA(Benjamini and Hochberg)] revealed that tomato plant geneexpression was more similar among the plants fed on byablated caterpillars and non-wounded plants than it was be-tween either herbivory treatments (Fig. 1). Further analysis ofthe heat map shows that ~50 % of the genes were expressed ina different direction or magnitude for the mock-ablated vs.ablated caterpillars.

J Chem Ecol

QPCR Verification of Follow-Up Caterpillar HerbivoryExperiments Correlate with Microarray Analysis The dif-ferential gene expression from the herbivory microarrayexperiment was consistent with qPCR verification (Figs. 2and 3). Of the 14 tomato genes designated as herbivore- andabiotic-response-related genes, nine had notably higher lev-els of expression for caterpillars with labial salivary glandscompared to the ablated treatment. They were abscisic acid-inducible dehydrin TAS14, acidic endochitinase, acid phos-phatase, allene oxide cyclase, arginase, PPO F, protease in-hibitor 2 CEV 1 and 2, and wound-induced protease inhibitorII. Only the expression of polyphenol oxidase D (PPO D) washigher for the ablated treatment.

A second experiment comparing tomato plant expres-sion was performed with the mock-ablated vs. ablatedcaterpillars, but examined at 12 hr post-herbivory. Simi-larly, there were trends of higher levels of gene expression fordehydrin, acidic endochitinase, arginase, pathogenensis-related protein PR-1A, threonine deaminase, and wound-induced protease inhibitors I and II for the caterpillarswith labial salivary glands versus the ablated treatment(Figs. 4 and 5).

Overall Genomic Expression of Mechanically Wounded To-mato Leaves In these experiments, tomato plants were

wounded with scissors (simulated herbivory) and treatedwith H. zea labial salivary gland extract, autoclaved salivarygland extract, or water, or left non-wounded, and harvested4 hr after treatment. Using the Benjamin and Hochbergmultiple test correction (adjusted P<0.05) we found 249ESTs that had significant differences in the expression levelsamong the treatments. Of these 249 ESTs, nearly half wereunknowns (Supplemental File 3). Approximately half ofthese ESTs were either up-regulated or down-regulated ingeneral between the mechanically wounded and the non-wounded control. Also, many of the ESTs that were simi-larly up-regulated among the wounded treatments, were inthe categories of anti-herbivore/anti-nutritive plant defensessuch as protease inhibitors, polyphenol oxidase, and threo-nine deaminase. Many of the ESTs that were comparablydown-regulated among the wounded treatments were gen-erally photosynthesis- and growth-related genes (Supple-mental File 3). The notable ESTs that were typicallystimulated more so by labial salivary gland extract includedabiotic, defense, and pathogen responses such as arginase,allene oxide cyclase, lipases, dehydrins, aquaporin 2, path-ogenesis protein 1 precursor, and transcription factor Pti4(Table 4). There also were numerous ESTs with unknownfunctions. As before, the q-values were determined with theStorey Method (Storey and Tibshirani, 2003). Using a less

Table 1 Primers used in the real-time quantitative PCR reactions

Sequence IDa Gene name and gene abbreviations Forward primer sequence Reverse primer sequence

SGN-U147451 Abscisic acid inducible dehydrin (DEH) 5!-TGCGCAAGACTGATGAATATG-3! 5!-TACCAGTACCCATGCCTTGAG-3!

SGN-U145331 Acid phosphatase (AcPho) 5!-TTAATGCGTGGATAGCTGAGG-3! 5!-TATAAGTGCTGCCCAGTTGCT-3!

SGN-U144297 Acidic 26 kDa endochitinase precursor (AcEc) 5!-AACTACGAACGAGCTGGACAA-3! 5!-ATAACGTTGTGCCATGATGGT-3!

SGN-U579208 Actin 5!-CCCAGAGGTACTCTTCCAACC-3! 5!-AAGCAGTGATTTCCTTGCTCA-3!

SGN-U147422 Allene oxide cyclase (AOC) 5!-CAGCTTCTACTTCGGCGATTA-3! 5!-ATTTTCACTTGACCGGAAACC-3!

SGN-U143272 1-Aminocyclopropane-1-carboxylate oxidase (ACC) 5!-CAATCCAGGAAGTGATGCAGT-3! 5!-GATCACTTTCCATTGCCTTCA-3!

SGN-U145219 Arginase (ARG) 5!-ACTGCAGGTCACTTGGACATC-3! 5!-CAAGGTGATATCGTTGGTGCT-3!

SGN-U566206 Lipoxygenase (LIP) 5!-GTTGCAGGGATAATGAGAGG-3! 5!-TCAGGCATCCTAATTGATGC-3!

SGN-U144656 Pathogenesis-related protein 1A1 precursor (PR-1A1) 5!-GTGGGACGATGAGAAGCAAT-3! 5!-CCGACTTACGCCATACCACT-3!

SGN-U143238 Pathogenesis-related protein 4 precursor (PR-4) 5!-TGTCCGAGAGGCCAGACTAT-3! 5!-ATGAACCACCACCCATTGTT-3!

AK323316.1 Peroxidase (POD) 5!-TGGCTCCAACAATAACATTCC-3! 5!-GTCAACCAGATCAAGCCCTTT-3!

SGN-U155880 Phenylalanine ammonia lyase (PAL) 5!-GGATCCATTGAACTGGGAAAT-3! 5!-ACCTTTGCAGCTACTGAAGCA-3!

Z12836.1 Polyphenol oxidase D (PPO D) 5!-TGCCACATGTTCATAGAGCTG-3! 5!-CCGCAATTGTGTCTTCATCTT-3!

SGN-U143365 Polyphenol oxidase F (PPO F) (Catechol oxidase) 5!-TACGCTGGCGATAACTGAACT-3! 5!-ATGGATACACCTTCACCACCA-3!

AK247244.1 Proteinase inhibitor 1 precursor(PI2Cev1) (ethylene-responsive)

5!-ATGTGGGCATCAAAGTTGTGT-3! 5!-GAGAGGAGCCAAGTCTCCAAT-3!

SGN-U143332 Proteinase inhibitor 2 (PI2Cev2)(ethylene-responsive)

5!-GTGAAGGAACGTCTGATCCAA-3! 5!-TTCCTTCTGAACGTGGACACT-3!

AK321298.1 S-adenosyl-L-methionine:salicyclic acidcarboxyl methyltransferase (SAMMT)

5!-AGTGGTGTGGCTGGTTCATT-3! 5!-CACTTGGTGGACTTGTACTTGC-3!

SGN-U145184 Systemin (SYS) 5!-GGAACATGAGGAGGGAGGATA-3! 5!-TGCATCATCATCTCCTTCAAT-3!

SGN-U143321 Threonine deaminase (ThrD) 5!-GGTTACGTTCAGGCAAAGAGG-3! 5!-CCCACAAGTCCAACAAATGTC-3!

SGN-U143220 Vacuolar proton ATPase proteolipid subunit (VATPS) 5!-TGCTCACCTCTCATCTGGTCT-3! 5!-AGCTCTAACACCAGCATCACC-3!

SGN-U143556 Wound-induced proteinase inhibitor I precursor (WIN1) 5!-GTGATGGGCCAGAAGTCATAA-3! 5!-AAATCAGCTGGGACTGGAGAG-3!

SGN-U143329 Wound-induced proteinase inhibitor II precursor (WIN2) 5!-TATTTGCCTTGGGTTCATCAC-3! 5!-CACAGGGTACAAGGGTTGCTA-3!

a Sol Genomics Network Unigene ID (http://solgenomics.net) or NCBI Accession ID (http://www.ncbi.nlm.nih.gov)

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conservative FDR of 4.64 % than the Benjamin-Hochbergmethod a total of 560 ESTs were significantly different intheir expression (Supplemental File 4). This larger set

included an additional 176 ESTs over the 384 determinedwith the Benjamin-Hochberg method and could be usefulfor further review.

Table 2 Relative fold difference of tomato leaf gene expression from 24 hr of herbivory by caterpillars with (Mock) or without (Ablated) labialsalivary glands vs. non-wounded (NW) plants analyzed with microarrays (P<0.05; HSD)

Putative annotation and function Treatments and fold difference

Plant Defense and Abiotic Resistance

Gene ID Annotation NW Mock Ablated

SGN-U143272 1-aminocyclopropane-1-carboxylate oxidase 1.00(a) 2.54(b) 1.42(c)

SGN-U147451 abscisic acid (dehydrin TAS14) 1.00(a) 7.00(b) !1.11(a)

SGN-U145331 acid phosphatase 1.00(a) 12.29(b) 3.12(c)

SGN-U143488 aminopeptidase 1, Leucine aminopeptidase 1.00(a) 6.39(b) 2.04(ab)

SGN-U145219 arginase [Arabidopsis thaliana] 1.00(a) 8.82(b) 5.59(b)

SGN-U143342 aspartic protease inhibitor 1 precursor 1.00(a) 3.96(b) 2.03(c)

SGN-U144440 beta-amyrin synthase (triterpene synthesis) 1.00(a) 2.61(b) !2.82(c)

SGN-U143930 bifunctional lysine-ketoglutarate reductase (lysine regulation) 1.00(a) 2.98(b) !1.46(a)

SGN-U147604 copper amine oxidase-like protein 1.00(a) 7.59(b) 1.44(a)

SGN-U145112 cytosol ic cysteine synthase (heavy metal tolerance) 1.00(a) 4.83(b) !1.70(a)

SGN-U144134 dehydrin 2 1.00(a) 2.27(b) !1.12(a)

SGN-U144132 dehydrin homolog CI7 1.00(a) 3.01(b) !1.13(a)

SGN-U144127 ethylene-responsive proteinase inhibitor I 1.00(a) 50.29(b) 21.64(b)

SGN-U145376 geranyl diphosphate synthase (terpene synthesis) 1.00(a) 1.85(b) !1.91(c)

SGN-U144943 kunitz-type enzyme inhibitor S9C11 1.00(a) 3.77(b) 1.52(c)

SGN-U153466 multicystatin precursor 1.00(a) 6.06(b) 2.35(c)

SGN-U150123 multicystatin; cystatin 1.00(a) 7.71(b) 2.14(c)

SGN-U143365 polyphenol oxidase F 1.00(a) 62.46(b) 45.95(c)

SGN-U143905 proteinase inhibitor type II 1.00(a) 7.75(b) 2.98(c)

SGN-U143332 proteinase inhibitor type II CEVI57 1.00(a) 18.85(b) 6.23(c)

SGN-U143803 putative gamma TIP (aquaporin related) 1.00(a) !2.73(b) 1.06(a)

SGN-U143321 threonine deaminase 1.00(a) 17.04(b) 4.35(c)

SGN-U143556 wound-induced proteinase inhibitor I 1.00(a) 342.62(b) 161.41(c)

SGN-U143329 wound-induced proteinase inhibitor II 1.00(a) 208.79(b) 53.90(c)

Hypersensitive Response Regulation, Reactive Oxygen Species Regulation, and Pathogen related proteins

SGN-U144297 acidic 26 kDa endochitinase precursor 1.00(a) 3.19(b) !2.04(c)

SGN-U150426 anthranilate phosphoribosyltransferase, putative 1.00(a) 2.10(b) !1.41(a)

SGN-U150956 beta-carbonic anhydrase 1.00(a) 8.86(b) !1.31(a)

SGN-U144188 DIP-1 1.00(a) 3.72(b) 1.52(a)

SGN-U143206 glyceraldehyde 3-phosphate dehydrogenase A 1.00(a) !2.80(b) 1.08(a)

SGN-U143179 lipid transfer protein 2 1.00(a) 2.08(b) !1.37(a)

SGN-U143485 metallothionein-like protein - tomato 1.00(a) 1.72(b) !1.41(a)

SGN-U143242 pathogenesis-related leaf protein 6 1.00(a) 2.28(b) 1.25(a)

SGN-U161489 pathogenesis-related protein P2 precursor 1.00(a) 1.38(b) !2.00(c)

SGN-U143410 phytoene synthase 1, biosynthesis carotenoids 1.00(a) 4.97(b) !1.27(a)

SGN-U147563 polyamine oxidase 1.00(a) !3.10(b) 1.05(a)

SGN-U143309 secretory peroxidase 1.00(a) 1.57(b) !2.03(c)

SGN-U144153 serine carboxypeptidase 1.00(a) !2.64(b) 1.43(a)

SGN-U143744 translation-inhibitor protein 1.00(a) 17.92(b) 2.93(c)

Gene ID0SOL Genomics Network Unigene ID; different letters0significant difference between treatments for each gene at P<0.05

J Chem Ecol

QPCR Verification of Mechanically Wounded Tomato LeavesCorrelates with Microarray Analysis For the mechanicallywounded tomato plant microarray experiment gene expres-sion, qPCR analysis was performed for 12 ESTs that weredesignated as herbivore- and abiotic-related. In addition tothe wounded tomato leaves described above, we also exam-ined glucose oxidase, which is a component of labial sali-vary gland extract, as an additional treatment. In all cases,the qPCR results correlated with the microarray results. Ofthese genes that were investigated, a handful of cases werestimulated more so by labial salivary gland extract after only4 hr, such as acidic endochitinase, allene oxide cyclase,arginase, and lipoxygenase (Figs. 6 and 7). For the GOXapplication on wounded tomato leaves, several of thesegenes such as PPO F, wound-induced protease inhibitor 2,protease inhibitor 2 CEV 2, and threonine deaminase werestimulated at an even higher level by GOX. However, forthe remaining wounded treatments the expression of theseplant defenses was not markedly different.

Discussion

Plants can discriminate between various types of herbivorybeyond mere physical wounding. Much of this ability comesfrom the application of oral secretions of insect herbivoresonto plant wounds during feeding (Felton, 2005; Korth andThompson, 2006; Mithöfer and Boland, 2008). However, mostcaterpillar HAMPs studies have focused on regurgitant despitethe extremely low levels of regurgitant secreted during herbiv-ory by some caterpillars (Peiffer and Felton, 2005). Regurgitantis of mixed origin and can consist of labial and mandibularsaliva, gut contents including partially digested plant tissues,gut compounds, gut enzymes, and gut microbials.

In this study, we examined the effect of H. zea caterpillarlabial saliva on the transcriptome profile of tomato plants. Therole of caterpillar labial saliva in eliciting plant defenses has

been partially elucidated with the use of surgeries to eithercauterize the caterpillar’s spinneret or to remove the caterpil-lar’s labial salivary glands (Musser et al., 2002a, 2005a, 2006;Bede et al., 2006; Delphia et al., 2006; Weech et al., 2008). Wecompared the expression of several thousand genes of S. lyco-persicum plants wounded by H. zea caterpillars with (mock-ablated) or without (ablated) labial salivary glands. In addition,we mechanically wounded tomato leaflets with scissors andpainted labial salivary gland extract, autoclaved labial salivarygland extract, water, or glucose oxidase, a component of labialsaliva. Based on Peiffer and Felton (2005), a 0.5ug of GOX isapplied per hour by H. zea caterpillars, so we roughly appliedthe equivalent of 10 to 20 hr of salivary secretions for eachleaflet wounded. We determined that caterpillar labial salivahas the ability to alter tomato plant gene expression beyondmere physical damage in both experiments. The levels of geneexpression of numerous genes correlated between actual cater-pillar herbivory and tomato plants mechanically wounded andtreated with labial salivary glands or GOX. Tomato plant geneexpression influenced by labial salivary gland extracts includeabscisic acid-inducible dehydrin TAS14, acidic endochitinase,acid phosphatase, allene oxide cyclase, arginase, polyphenoloxidase, threonine deaminase, an assortment of protease inhib-itors, and plant pathogen-related proteins. Herbivory by mock-ablated caterpillars generally elicited higher levels of tomatoplant gene expression compared to herbivory by ablated cater-pillars. Additionally, caterpillar labial saliva stimulated genesfrom various plant physiological categories, particularlyabiotic-related, anti-herbivore/anti-nutritive-related, and hyper-sensitive/pathogen-related genes. The expression of genes thatwere down-regulated more so by mock-ablated caterpillarscompared to ablated caterpillars was photosynthesis-relatedand several hypersensitive/pathogen-related genes.

Caterpillar Saliva Alters Plant Defense-Related Genes Theexpression of numerous anti-herbivore/anti-nutritive plantdefense genes was stimulated by the caterpillar herbivory

Table 3 Relative fold differenceof tomato leaf gene expressionfrom 24 hr of herbivory bycaterpillars with (Mock) orwithout (Ablated) labial salivaryglands vs. non-wounded (NW)plants analyzed with microarrays(P<0.05; HSD)

Gene ID0SOL Genomics Net-work Unigene ID; differentletters0significant differencebetween treatments for eachgene at P<0.05

Putative annotation and function Treatments and fold difference

Photosynthesis

Gene ID Annotation NW Mock Ablated

SGN-U150956 beta-carbonic anhydrase 1.00(a) 8.86(b) !1.31(a)

SGN-U143631 Carbonic anhydrase 1.00(a) !2.17(b) !1.28(ab)

SGN-U155551 Photosystem I reactioncenter subunit IV B

1.00(a) !3.57(b) !1.85(ab)

SGN-U143798 Photosystem II 10 kDa polypeptide 1.00(a) !3.03(b) !2.08(b)

SGN-U143215 Photosystem II 22 kDa protein 1.00(a) !4.00(b) !2.08(ab)

SGN-U152300 Plastocyanin, chloroplast precursor 1.00(a) !4.55(b) !1.32(a)

SGN-U143464 Ribulose bisphosphate carboxylase/oxy. 1.00(a) !5.26(b) !2.32(c)

J Chem Ecol

treatments in comparison to the non-wounded control.Mock-ablated caterpillars tended to quantitatively stimulatehigher levels of expression of plant defense genes (e.g.,protease inhibitors, polyphenol oxidase, threonine deami-nase) than ablated caterpillars. While the expression ofmany of the genes was nearly equally stimulated by themechanically wounded plants, GOX tended to stimulatethese plant defenses higher for arginase, polyphenol oxi-dase, threonine deaminase, and wound inducible/proteaseinhibitors II. These results were similar to a recent studyby Felton’s lab that showed increases in protease inhibitor

Mock NW Ablated

-2 0 2

Fig. 1 Cluster analysis of tomato gene expression from 24 hr herbiv-ory by caterpillars with labial salivary glands (Mock) vs. caterpillarswithout labial glands (Ablated) compared to non-wounded controls(NW). Genes that appear redder are up-regulated. Genes that appearbluer are down-regulated

*

*

*

N=3

Rel

ativ

e F

old

Cha

nge

Fig. 2 Relative fold differences of induced plant defenses 24 hr afterwounding on tomato leaves determined with qPCR. Tomato planttreatments were non-wounded plants (NW), mock-ablated caterpillars(MOCK) with intact labial salivary or caterpillars with ablated labialsalivary gland extracts (ABL). Genes of interest included: VATPS0Vacuolar proton ATPase proteolipid subunit; ACC01-aminocyclopro-pane-1-carboxylate oxidase; PAL0phenylalanine ammonia lyase;ARG0Arginase; PPOf0Polyphenol oxidase f; PPOd0Polyphenol ox-idase d; SYST0Systemin; APHOS0Acid phosphatase; ACEC0Acidic26 kDa endochitinase precursor; and AOC0allene oxide cyclase.VATPS was used as a control for genes not altered in the microarrayanalysis. Asterisk represent tomato plant gene expression that wassignificantly different between mock-ablated caterpillars versus ablatedcaterpillar and the non-wounded treatment. All wounded treatmentswere significantly different from NW. Bars indicate means±S.E

*

N=3

Fig. 3 Relative fold differences of wound induced direct plantdefenses 24 hr after wounding on tomato leaves determined withqPCR. Tomato plant treatments were non-wounded plants (NW), cat-erpillars with labial salivary glands (MOCK), or caterpillars withoutlabial salivary gland (ABL). Genes of interest included: WIN10Wound-induced proteinase inhibitor I precursor; WIN20Wound-in-duced proteinase inhibitor II precursor; PI2CEV10Proteinase inhibitor1 precursor (ethylene-responsive); PI2CEV20Proteinase inhibitor 2precursor (ethylene-responsive); and DEH0Abscisic acid inducibledehydrin. Asterisk represent tomato plant gene expression that wassignificantly different between mock-ablated caterpillars versus ablatedcaterpillars and the non-wounded treatment. All wounded treatmentswere significantly different from NW. Bars indicate means±S.E

J Chem Ecol

and trichomes were due to H. virescens saliva that correlatedto GOX levels (Tian et al., 2012). It should be noted thatwhile caterpillar labial salivary gland extract has GOX, thelevels of pure GOX applied to the tomato leaves woundedwith scissors were two to four fold higher than the amountused in the labial salivary gland extracts (Musser et al.,

2006). We speculate that GOX has a concentration effecton tomato plant defenses that could be seen in actual cater-pillar herbivory or with the application of higher amounts oflabial salivary gland extract. Albeit, there are likely otherfactors in labial saliva that mediate GOX’s effect on tomatoplants, with recent studies having shown substantial levels

N=6

*

Fig. 4 Relative fold differences of wound induced direct plantdefenses 12 hr after wounding determined with qPCR. Tomato planttreatments were non-wounded plants (NW), caterpillars with labialsalivary glands (MOCK), or caterpillars without labial salivary glands(ABL). Genes of interest included: AcEc0acidic 26 kDa endochiti-nase; AOC0allene oxide cyclase; Arg0arginase, Pal0phenylalanineammonia lyase, PR1A10pathogenesis-related protein 1A1 precursor,

and Sammt0S-adenosyl-L-methionine:salicyclic acid carboxyl meth-yltransferase. Asterisk represent tomato plant gene expression that wassignificantly different between mock-ablated caterpillars vs. ablatedcaterpillars and the non-wounded treatment. All wounded treatmentswere significantly different from NW except AcEc and Sammt. Barsindicate means±S.E

*

N=6

Fig. 5 Relative fold differences of wound induced direct plantdefenses 12 hr after wounding determined with qPCR. Tomato planttreatments were non-wounded plants (NW), caterpillars with labialsalivary glands (MOCK), or caterpillars without labial salivary glands(ABL). Genes of interest included: DEH0Abscisic acid inducibledehydrin; PI2CEV20Proteinase inhibitor 2 precursor (ethylene-re-sponsive); PPOf0Polyphenol oxidase f; ThrD0Threonine deaminase;

WIN10Wound-induced proteinase inhibitor I precursor; and WIN20Wound-induced proteinase inhibitor II precursor. Asterisk representtomato plant gene expression that was significantly different betweenmock-ablated caterpillars vs. ablated caterpillars and the non-woundedtreatment. All wounded treatments were significantly different fromNW. Bars indicate means±S.E

J Chem Ecol

of fructosidases, carboxylesterase, and ecdysone oxidase(Celorio-Mancera et al., 2011; Tian et al., 2012).

The wound-inducible proteinase inhibitors are plantdefenses that decrease the digestibility of the leaf material byinhibiting digestive proteases such as animal/insect endopep-tidases, chymotrypsin and trypsin, thus making the plant lesspalatable (Green and Ryan, 1972; Graham et al., 1985). Cat-erpillars with salivary secretions tended to increase proteinaseinhibitor expression compared to ablated caterpillars. Like-wise application of GOX onto damaged tomato leaves dra-matically increased some protease inhibitor levels, more sothan the other wounded treatments. This suggests that thisexpression may be due in part to the salivary enzyme GOX.

Orozco-Cárdenas et al. (2001) showed that GOX in the pres-ence of glucose stimulated protease inhibitors in tomato plantsmore so than in plants not treated with GOX. Larvae from thesister species H. armigera and H. assulta stimulated similarplant defense compounds, but at differing intensity comparedto the expression induced by S. litura, partly due to varyinglevels of salivary GOX (Zong andWang, 2004). However, theeffects of labial saliva GOX on tomato plant defenses may beshort lived, as GOX may be down-regulated in caterpillarsaliva after the larvae have fed on tomato plants, and it hasbeen shown that GOX levels are diet-dependent (Peiffer andFelton, 2005; Babic et al., 2008). Other studies have demon-strated that caterpillar herbivory stimulates protease inhibitors,

Table 4 Microarray Analysis Experiments- Relative ratio of selected tomato leaves genes up- or down-regulated by wounding with scissors andtreated with labial salivary gland extract, autoclaved labial salivary gland extract or water vs. non-wounded tomato leaves (P<0.05; HSD)

Putative annotation and function Treatments and fold difference

Plant Defense and Abiotic Resistance

Gene ID Annotation NW S. ext. Auto Water

SGN-U143272 1-aminocyclopropane-1-carboxylate oxidase 1.00(a) 2.23(b) 2.57(b) ~

SGN-U147451 abscisic acid inducible dehydrin 1.00(a) 1.99(b) 1.29(a) !1.66(a)

SGN-U145331 acid phosphatase 1.00(a) 4.15(b) 7.15(b) ~

SGN-U144297 acidic 26 kDa endochitinase precursor 1.00(a) 5.97(b) 2.78(c) ~

SGN-U147422 allene oxide cyclase 1.00(a) 4.57(b) 2.81(ab) 1.58(a)

SGN-U143488 aminopeptidase 1, Leucine aminopeptidase 1.00(a) 2.48(b) 2.81(b) 1.43(a)

SGN-U143967 aquaporin 2 1.00(a) 1.99(b) 1.15(a) !1.47(a)

SGN-U145219 arginase [Arabidopsis thaliana] 1.00(a) 5.59(b) 3.34(c) 4.49(bc)

SGN-U143342 aspartic protease inhibitor 1 precursor 1.00(a) !1.07(a) 1.12(a) !1.01(a)

SGN-U143809 cinnamic acid 4-hydroxylase 1.00(a) 4.04(b) 4.63(b) 5.83(b)

SGN-U144134 dehydrin 2 1.00(a) 2.31(b) 1.73(c) 1.31(a)

SGN-U144127 ethylene-responsive proteinase inhibitor I 1.00(a) 2.15(b) 1.97(b) 2.11(b)

SGN-U150123 multicystatin; cystatin 1.00(a) 2.57(b) 2.27(b) 3.09(b)

SGN-U145477 peroxidase 1.00(a) !2.86(a) !2.56(a) !1.93(a)

SGN-U155880 phenylalanine ammonia-lyase 1.00(a) 4.11(b) 4.47(b) 8.60

SGN-U143365 polyphenol oxidase F 1.00(a) 3.95(b) 4.04(b) 4.34(b)

SGN-U149540 protein phosphatase 2C 1.00(a) 2.62(b) 2.32(b) 6.82

SGN-U143905 proteinase inhibitor type II 1.00(a) 1.95(a) 1.51(a) 2.18(a)

SGN-U143332 proteinase inhibitor type II CEVI57 1.00(a) 2.13(a) 1.60(a) ~

SGN-U148267 subtilisin-like proteinase 1.00(a) 6.16(b) 6.60(b) 5.84(b)

SGN-U143321 threonine deaminase 1.00(a) 3.08(b) 3.33(b) 3.78(b)

SGN-U143556 wound-induced proteinase inhibitor I 1.00(a) 3.66(b) 3.21(b) 3.46(b)

SGN-U143329 wound-induced proteinase inhibitor II 1.00(a) 4.01(b) 3.55(b) 3.64(b)

SGN-U145296 Xaa-Pro aminopeptidase 2 1.00(a) 3.69(b) 3.55(b) 3.91(b)

Pathogenesis related proteins

SGN-U150244 mads box protein TDR3 - tomato 1.00(a) 9.71(b) 9.97(b) 9.14(b)

SGN-U143838 pathogenesis-related protein PR-1 1.00(a) 1.28(a) 1.73(a) !3.31(b)

SGN-U144888 pto-responsive gene 1 protein 1.00(a) 3.68(b) 4.80(b) 4.25(b)

SGN-U146226 transcription factor Pti4 - tomato (fragment) 1.00(a) 1.81(b) 2.63(b) !25.38(c)

SGN-U143744 translation-inhibitor protein 1.00(a) 4.29(b) 4.28(b) 2.36(b)

Gene ID0SOL Genomics Network Unigene ID; different letters0significant difference between treatments for each gene at P<0.05

J Chem Ecol

but the response can be attenuated by the presence ofsalicylate-inducing aphids (Rodriguez-Saona et al., 2010).Spodoptera exigua caterpillar labial saliva suppresses theinduction of A. thaliana trypsin inhibitor most likely through

the interference of the jasmonate pathway and activation ofthe SA pathway (Weech et al., 2008). Our data support theconclusion that caterpillar labial saliva alters plant gene ex-pression in the tomato; H. zea response and GOX are likelyinvolved in these differences.

Allene oxide cyclase (AOC) is critical for the synthesis ofjasmonate, which regulates anti-nutritive defenses and hasbeen shown to be stimulated by herbivory. Herbivory by themock-ablated or ablated caterpillars stimulates allene oxidecyclase expression in comparison to the non-wounded plants.There was a higher trend of AOC expression for the mock-ablated caterpillars after 24 hr in the qPCR results compared toresults for the ablated caterpillars. Tomato plants that weremechanically wounded with scissors after only 4 hr stimulatedAOC in comparison to non-wounded plants. Interestingly,wounded leaves painted with labial salivary gland extract hadthe highest levels of AOC in comparison to wounded plantstreated with autoclaved labial saliva or water. This suggests thatH. zea labial saliva may enhance jasmonate synthesis, partic-ularly during early events of herbivory. Ethylene is synthesizedfrom the enzymatic activity of 1-aminocyclopropane-1-carbox-ylate synthase and 1-aminocyclopropane-1-carboxylate oxi-dase (ACC) (Adams and Yang, 1979; Yang and Hoffman,1984; Liu et al., 1985; O’Donnell et al., 1996). Here, weshowed that both caterpillar herbivory treatments induced sig-nificant levels of ACC expression compared to non-woundedplants. However, mock-ablated caterpillars tended to increaseplant ACC levels only slightly more than ablated caterpillars.

Polyphenol oxidase forms quinones that cause reactionsthat bind up plant proteins, making them less digestible for thecaterpillar herbivore. We found significantly higher levels ofPPO F gene expression in wounded leaves. Mock-ablated

*

*

N=3

Fig. 6 Relative fold differences of wound induced direct plantdefenses 4 hr after wounding determined with qPCR. Tomato planttreatments were non-wounded plants (NW) or wounded and treatedwith labial salivary gland extract (SAL), autoclaved labial salivarygland extract (AUTOSAL), water (H2O) or glucose oxidase (GOX).Genes of interest included PAL0phenylalanine ammonia lyase; AOC0allene oxide cyclase; LIP0Lipoxygenase; ACEC0Acidic 26 kDaendochitinase precursor; VATPS0Vacuolar proton ATPase proteolipidsubunit. VATPS was used as control for genes not altered in themicroarray analysis. Asterisk represent tomato plant gene expressionthat was significantly different between SAL compared to AUTOSAL,GOX and H2O. All wounded treatments were significantly differentfrom NW. Bars indicate means±S.E

N=3

Fig. 7 Relative fold differences of induced plant defenses 4 hr afterwounding determined with qPCR. Tomato plant treatments were non-wounded plants (NW) or wounded and treated with labial salivary glandextract (SAL), autoclaved labial salivary gland extract (AUTOSAL),water (H2O) or glucose oxidase (GOX). Genes of interest included:PPOf0polyphenol oxidase f; PPOd0polyphenol oxidase d; WIN10

wound-induced proteinase inhibitor I precursor; WIN20wound-inducedproteinase inhibitor II precursor, PI2CEV20proteinase inhibitor I precur-sor (ethylene-responsive); PI2CEV20proteinase inhibitor 2 precursor(ethylene-responsive); ARG0arginase; and ThrD0threonine deaminase.All wounded treatments were significantly different from NW. Barsindicate means±S.E

J Chem Ecol

caterpillars with labial saliva generally stimulated PPO Fmoreso than ablated caterpillars. Likewise, mechanically woundedtomato plants painted with GOX stimulated higher levels ofPPO F than the other similarly wounded tomato leaves. Cor-roborating evidence shows that H. armigera with higher lev-els of GOX stimulated higher levels of PPO in tobacco plantsthan H. assulta with negligible levels of GOX (Zong andWang, 2007). This may be due to the effect of hydrogenperoxide (H2O2) from labial saliva GOX activity, similar tothe increase in PPO expression in response to tissue damage,and bacterial and fungal pathogen infection (Constabel et al.,1995; Thipyapong and Steffens, 1997). In addition, the ex-pression of other tomato plant anti-nutritive defenses such asarginase and threonine deaminase were stimulated by H. zeaherbivory and was induced slightly more so by mock-ablatedcaterpillars than ablated caterpillars. Arginase also was stim-ulated at higher levels in mechanically wounded tomatoleaves treated with labial salivary gland extract or GOX com-pared to the other wounded plants. Both arginase and threo-nine deaminase are plant defenses that catabolize the aminoacids before they are absorbed by the insect herbivore (Felton,2005). Arginase is a JA-inducible protein that catabolizessingle amino acid molecules of arginine in the herbivore (M.sexta) midgut (Chen et al., 2005), preventing the metabolismof essential amino acids and thus limiting the herbivore’sgrowth.

Caterpillar Saliva Alters Abiotic-Related Stress Genes Thetomato plant gene expression of a variety of dehydrins, heatshock proteins, aquaporins, and heavy metal toleratinggenes were notably altered by herbivory from mock-ablated caterpillars with labial salivary secretions comparedto the ablated caterpillars. Similarly, tomato plants mechan-ically wounded with scissors and painted with labial sali-vary gland extracts had substantially higher levels of theexpression of some of these genes, particularly dehydrins,compared to similarly wounded tomato leaves. Abscisicacid-related dehydrin TAS14 is an abscisic acid- anddrought-related gene that was significantly stimulated bysaliva in the mock-ablated treatment compared to the ablat-ed treatment. Dehydrin expression changes in response tocold, freezing, drought, and abscisic acid, and can providestability in cellular structures (Heiden et al., 1999; Borovskiiet al., 2002; Gao et al., 2008; Bae et al., 2009). Pieris rapaecaterpillar herbivory stimulates water-stress-related proteinexpression in Arabidopsis plants compared to just mechan-ically wounded leaves (Reymond et al., 2000). Dehydrinexpression also is induced by mechanical wounding, JA,and methyl jasmonate in some species (e.g., Picea glauca,Richard et al., 2000), but not by wounding in other species(e.g., hybrid poplar, Bae et al., 2009). Here, we showed thatcaterpillar labial saliva affects regulation of the expressionof abiotic-related plant responses.

Caterpillar Saliva Alters Hypersensitive/Pathogen-RelatedGenes The gene expression of a wide range of hypersensitive/pathogen-related genes such as acidic endochitinase, beta-carbonic anhydrase, polyamine oxidase, DIP-1, PR-6, phy-toene synthase 1, PR-1, and translation-inhibitor protein werestimulated more so by caterpillars that could secrete labialsaliva than those caterpillars with ablated salivary glands.These results support the idea that caterpillar labial salivamay stimulate aspects of the salicylate pathway. This induc-tion is due possibly to the activity of salivary enzyme GOX inthe production H2O2. A similar effect was demonstrated in theinduction of tobacco plant defenses from herbivory by S.exigua, which has GOX in its oral secretions (Diezel et al.,2009). Earlier studies have suggested that caterpillar labialsaliva could interfere with the jasmonate pathway and stimu-late components of systemic acquired resistance (SAR) path-way [H. zea effects on tobacco and tomato (Musser et al.,2005a) and S. exigua on Arabidopsis (Weech et al., 2008)].Utilizing Western blot analysis, Musser et al. (2005a) showedthat pathogenesis-related protein 1a was stimulated by H. zealabial salivary gland extract with glucose oxidase, vs. labialsalivary gland extract in which GOXwas removed. Therefore,caterpillars that lack GOX may not stimulate the salicylatepathway (Diezel et al., 2009). Here, we showed that H. zealabial saliva that contains GOX does stimulate the expressionof genes commonly associated with plant pathogen defenses.

Acidic endochitinase was stimulated at a higher level bymock-ablated caterpillars compared to ablated caterpillars. Wedetermined that mechanically wounded tomato plants paintedwith labial gland extract also had a higher level of acidicendochitinase. Acidic endochitinase is a chitin-degrading plantdefense enzyme. Chitin is found in fungal cell walls and theinsect exoskeleton and gut lining (Yeboah et al., 1998).Chitinases can be induced by both ethylene and salicylic acid.Chitinase expression does not change in response to woundingby ablated caterpillars; however, it is significantly increased byherbivory from caterpillars secreting saliva (Tables 2 and 4).This up-regulation of products of the SA pathway is likely dueto the presence of H2O2 resulting from GOX activity in H. zealabial saliva, as H2O2 increases are known to stimulate SA(Leon et al., 1995).

Caterpillar Saliva Alters Photosynthesis-Related Genes Ingeneral, while herbivory reduced the expression of photosyn-thesis genes compared to non-wounded plants, herbivoryfrom mock-ablated caterpillars further reduced the expressionof tomato photosynthesis-related genes compared to herbivo-ry from the ablated caterpillars. The production of plantdefenses is costly to the plant. As direct and indirect anti-herbivore defenses are mounted to protect against damage,other areas of plant physiological processes are reduced.

In Summary, we demonstrated a pattern of both stimula-tory and inhibitory effects of caterpillar saliva on plant

J Chem Ecol

responses. Unlike most other studies that use caterpillarregurgitant, here, we focused on labial saliva, and excludedany plant-derived compounds present in the foregut, gutenzymes, and other gut or bacterial compounds that couldalso affect plant gene expression. We present a caterpillarsaliva-manipulated plant transcriptome, with a focus onabiotic-, defense-, and pathogen-related genes. Generallyspeaking, caterpillar saliva qualitatively stimulated moreabiotic-related (dehydrins, copper amine oxidase, cytosoliccysteine synthase) and pathogen-related genes (acidic endo-chitinase, beta-carbonic anhydrase, polyamine oxidase, DIP-1, PR-6, phytoene synthase 1, PR-1, translation-inhibitor pro-tein) than the ablated treatment. In general, anti-nutritive plantdefenses were stimulated by herbivory; however, the quanti-tative responses were typically higher for the caterpillars withlabial saliva compared to those for ablated caterpillars, inparticular affecting a variety of plant defenses such as acidphosphatase, cystatin, polyphenol oxidase, protease inhibi-tors, and threonine deaminase. While it is advantageous forthe plant to appropriately defend itself against specific threats,at the same time it is advantageous for the herbivore tominimize detection. These conflicting interests are reflectedby the complex pattern of plant responses. This study providesa robust set of tomato plant gene candidates whose expressionis specifically altered by H. zea caterpillar labial saliva forfuture investigations into herbivore-associated molecularpatterns.

Acknowledgements Funding was provided by the National ScienceFoundation Plant Genome Research Initiative (No. 0820367 to ROMand SMH), the National Research Initiative of the USDA CooperativeState Research, Education and Extension Service (No. 2004–01540 toROM and SMH) and the Western Illinois University research council.We thank Drs. Amy Patrick Mossman (WIU), Patrick Dowd (USDA-ARS, Peoria), and Eric T. Johnson (USDA-ARS, Peoria) for theirreview of this manuscript. We also thank the Genome Consortium forActive Teaching for assistance in obtaining microarray chips used byundergraduate students in this project.

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