Signal interactions in pathogen and insect attack: systemic plant-mediated interactions between pathogens and herbivores of the tomato,Lycopersicon esculentum

Physiological and Molecular Plant Pathology (1999) 54, 115–130 Article No. pmpp.1998.0193 available online at http :\\www.idealibrary.com on

Signal interactions in pathogen and insect attack : systemic plant-mediated interactions between pathogens and herbivores of the tomato, Lycopersicon esculentum M. J. S"*, A. L. F#†, S. S. D"‡ and R. M. B#§ " Department of Entomology and # Department of Plant Pathology, University of California, Davis, CA 95616, USA (Accepted for publication NoŠember 1998)

Plant-mediated interactions (i.e., induced resistance) between plant pathogens and insect herbivores were investigated using several pests of the cultivated tomato, Lycopersicon esculentum. Single leaflets of tomato leaves were injured by allowing a third-instar HelicoŠerpa zea larva to feed on the leaflets or by inoculating the leaflets with Pseudomonas syringae pv. tomato (the causal agent of bacterial speck in tomato ; Pst) or with Phytophthora infestans (the causal agent of late blight). Leaflets on separate plants were sprayed with benzothiadiazole, a chemical inducer of resistance to Pst. The effects of these treatments on the resistance of uninoculated or undamaged leaflets to both Pst and H. zea were then assessed after appropriate periods of time. The levels or activities of several defense-related proteins were determined in parallel. Infection of leaflets by Pst decreased the suitability of uninoculated leaflets of the same leaf for both H. zea and for Pst. Similarly, feeding by H. zea caused leaf-systemic increases in resistance to both H. zea and Pst. Infection of leaflets by P. infestans, in contrast, had no effect on resistance of leaflets to H. zea. Treatment of leaves with benzothiadiazole induced resistance to Pst but improved suitability of leaflets for H. zea. Feeding by H. zea caused the systemic accumulation of proteinase inhibitor mRNA and the systemic induction of polyphenol oxidase activity ; in contrast, treatment with benzothiadiazole and inoculation with P. infestans caused the systemic accumulation of pathogenesis-related protein mRNA and the systemic induction of peroxidase activity. Inoculation of leaflets with Pst caused the leaf-systemic accumulation of both pathogenesis-related protein and proteinase inhibitor mRNA and the systemic induction of both peroxidase and polyphenol oxidase activity. These results provide clear evidence for reciprocal induced resistance involving certain pathogens and arthropod herbivores of tomato. In addition, these results provide several insights into the integration and coordination of the induced defenses of tomato against multiple pests and suggest that the expression of resistance against some pests may compromise resistance to others. # 1999 Academic Press

* Present address : Department of Entomology, Lousiana State University, Baton Rouge, LA 70803, USA. † Present address : Section of Plant Biology, University of California, Davis, CA 95616, USA. ‡ Deceased. § To whom correspondence should be addressed. Abbreviations used in text : BTH, benzothiadiazole-7-carbothioic acid S-methyl ester ; IR, induced resistance ; P4, pathogenesis-related protein 4 ; PIN, proteinase inhibitor ; PR, pathogenesis related ; Pst, Pseudomonas syringae pv. tomato ; SAR, systemic acquired resistance. 0885–5765\99\030115j16 $30.00\0

# 1999 Academic Press

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INTRODUCTION

Plants must contend with numerous pathogens and arthropod herbivores that differ considerably with respect to type and severity of damage inflicted, intimacy of association with the plant, and sensitivity to various plant defense mechanisms. Different pests can attack simultaneously, requiring that effective plant defenses be both multifaceted and integrated. Defense against pathogens and arthropods is provided in part by secondary chemicals, including defense-related proteins. A single plant may contain dozens of secondary chemicals from disparate chemical classes [13]. They often deter the growth or feeding of, or are acutely toxic to, the pathogens and arthropods exposed to them, and their presence is often strongly associated with resistance to pathogens and arthropods [13, 31]. Insect feeding and pathogen infection often induce changes in plant chemistry that are correlated with increases in pest resistance [6, 27, 42, 48]. Despite the necessary unity of plant defense against pathogens and arthropods, our understanding of the specific defense mechanisms and their relative functional significance remains fragmented. The study of plant-pathogen interactions and the study of plant-herbivore interactions have progressed largely independently, and each discipline has its own methodologies, experimental approaches, and definitions [3]. Moreover, within each discipline, there has been a tendency to study interactions in a rather parochial fashion ; that is, most mechanistic studies, until quite recently, have investigated the relationship between a single pest and a single type of defense-related factor or response, ignoring on the one hand the presence of other defense-related factors and responses in the plant and, on the other hand, the potential influence of other types of pests, and the attendant signals they generate, against which the plant must protect itself [13, 29]. The integration and coordination of a plant’s defenses against its various pathogen and arthropod pests is thus an important but relatively neglected topic. Inducible responses to pathogens and arthropods, because their expression can be manipulated by the investigator, are particularly useful systems for studying the integration and coordination of plant defenses. The two best-characterized pathways of plant response to pests are the octadecanoid and salicylic acid pathways, which give rise to two integrated sets [48] of chemical responses [34–38]. The octadecanoid pathway, usually associated with feeding by chewing insects or similar physical trauma, involves jasmonic acid as an intermediate signal and culminates in the production of proteins such as polyphenol oxidase and proteinase inhibitors [36]. The salicylate pathway, usually associated with infection by pathogens, involves salicylic acid as an intermediary signal and leads to the production of a number of compounds, most prominently the pathogenesis-related (PR) proteins [35]. Although there is evidence that certain compounds produced as a result of the action of jasmonates or salicylates are inhibitory to both pathogens and insects [9, 44], it is the involvement of the octadecanoid pathway in induced resistance against insects and the involvement of the salicylate pathway in induced resistance against pathogens that have received the most attention. Apart from reports that activation of the salicylic acid pathway can interfere with jasmonate-related gene expression [11, 33], there is relatively little information on coordination and integration of these two pathways, in large part because these pathways are usually studied separately (but see ref. 37, 38).

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The tomato, Lycopersicon esculentum Mill., has been an important model for the study of inducible responses to both arthropods and pathogens. The chemical responses of tomato foliage following feeding by arthropods, as well as the increases in resistance to arthropods which attend these chemical changes, have been characterized [14, 21, 36, 40, 42, 43]. Similarly, chemical responses of tomato foliage to pathogens, and the increases in resistance to pathogens which accompany these responses, have been described [4, 8, 10, 15]. Although there is overlap in some responses to different pathogens and insects, many of these changes in chemistry and resistance are specific to a particular biotic challenge [41, 44]. In a companion paper [18], we described the effects of application to tomato leaves of chemical and biological agents that engage certain hallmark salicylate- and jasmonate-related responses. That study provides a framework for the experiments reported in this paper, in which we sought to assess the performance of pathogens and insects on tomato leaves that had been induced by various agents to express responses typical of salicylate- or jasmonate-treatment. Specifically, we investigated, in various combinations, the systemic effect of challenge by a necrotic local lesion-forming bacterial pathogen, Pseudomonas syringae pv. tomato (Okabe) Young, Dye and Wilkie (Pst), a hemibiotrophic fungal pathogen, Phytophthora infestans (Mont.) de Bary, and a leaf chewing caterpillar (Lepidoptera ; Noctuidae), the corn earworm, HelicoŠerpa zea Boddie, on the suitability of uninoculated or undamaged leaflets to subsequent challenge by H. zea or Pst. We also used applications of benzothiadiazole (BTH), a synthetic mimic of salicylic acid [20], to induce resistance to Pst ; the effect of this treatment was used as a standard for comparison with the effects of other treatments. Corresponding analyses of systemic induction of proteinase inhibitors, polyphenol oxidase, peroxidase, and a tomato PR-protein were conducted to assess relationships between induced resistance and the expression of defense-related marker proteins. MATERIALS AND METHODS

Plant, insect, and pathogen material Young, greenhouse-grown tomato plants (L. esculentum cv. New Yorker) were used for all experiments. Plants were grown in 4 inch pots in a soil mix containing sand, perlite, lava rock, compost and peat moss. The soil mix also contained a slow-release fertilizer (14–12–14 N-P-K). Plants were watered daily. Natural light was supplemented by a single 1000-watt sodium vapor lamp during a 16 h photoperiod. Plants were used for experiments when they possessed three fully-expanded leaves and a fourth expanding leaf (20–25 days after sowing). H. zea and Spodoptera exigua Hubner (beet armyworm) were obtained as eggs from the USDA (Stoneville, MS, USA) and reared on an artificial diet in a growth chamber illuminated with incandescent and cool white fluorescent lamps during a 16 h photoperiod. P. syringae pv. tomato (Pst) was isolated from field-grown tomatoes (isolate Pst23, gift of D. Cooksey, Department of Plant Pathology, UC Riverside). The bacterial isolate was stored until use in a glycerol\LB broth mixture at k80 mC. To prepare inoculum, cells from the glycerol mix were transferred onto King’s B medium and allowed to grow for 2–3 days. Bacteria were taken directly from these plates and suspended in water, and bacterial concentration was estimated by measuring absorbance at 620 nm.

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P. infestans (race 1) was originally isolated from tomato (gift of M. Coffey, Department of Plant Pathology, UC Riverside). The fungal isolate was maintained on rye seed agar at 18 mC. Interactions between Pst and H. zea To test the leaf-systemic effects of localized infection by Pst on resistance of tomato foliage to H. zea and Pst, four-leaf tomato plants were assigned in equal numbers to three treatment groups. Plants in the first group were subjected to a localized infection by Pst, and those in the second group were mock-inoculated (control group). Inoculations were confined to the terminal leaflet of the third leaf. Leaflets were inoculated by gently rubbing entire upper surfaces of leaflets with a cotton-tipped applicator saturated with a bacterial suspension (2i10) colony forming units ml−"). Typical bacterial speck lesions were usually evident within 3–4 days. Plants in the third group were treated with BTH by spraying entire third leaves until run-off with a 1n2 m solution of BTH (Novartis, Inc., Greensboro, NC, USA). Five days after inoculation and BTH treatment, leaflets of the third leaf (except the terminal leaflet) were used to assess the effects of treatment on suitability of the leaf for Pst and\or H. zea, and to assess chemical changes caused by the treatments. Chemical assays and bioassays used to assess suitability of leaflets for Pst and H. zea are described below. Three trials of this experiment were conducted. The effects of infection by Pst on growth rates of H. zea were tested in two of these trials, and the effects of prior infection on resistance to subsequent infection by Pst were tested in all three trials. Effects of Pst infection on enzyme activities and expression of PINII and P4 mRNA were investigated in all three trials. An analogous procedure was used to investigate the consequences of localized feeding by H. zea. Terminal leaflets of the third leaves of one group of plants were subjected to feeding by H. zea, another group was left undamaged, and the entire third leaves of a third group were sprayed until run-off with BTH (1n2 m). Plants were damaged with corn earworm larvae by confining fourth-instar larvae to terminal leaflets of third leaves using a clip cage and allowing larvae to feed for 8–12 h ; control plants received a clip cage with no larvae. Leaflets were used for assays of leaflet suitability and chemistry two days after damaging plants. Plants were treated with BTH three days prior to damaging plants (five days before assaying for resistance and chemical effects). Methods for bioassays and chemical assays are given below. Five trials of this experiment were conducted. The effects of H. zea feeding on resistance to H. zea was examined in three of the trials ; the effect of H. zea feeding on resistance to Pst was examined in four trials ; the chemical effects of H. zea feeding were investigated in three trials. Interactions between P. infestans and noctuids Three experiments similar to those described above were carried out to examine the effects of localized infection by P. infestans on the susceptibility of tomato leaves to noctuid larvae. Terminal leaflets of third leaves of a group of 8 to 12 plants were inoculated with P. infestans by gently applying with a paint brush to the upper sides of leaflets a sporangial suspension (10& spores ml−"), and terminal leaflets of third leaves

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of another set of plants were mock-inoculated with water. A BTH control was not included in these experiments. Plants of both treatments were placed in clear plastic boxes lined with wet paper towels to maintain humidity. The boxes were sealed and placed in a growth chamber providing a 16 h photoperiod at 18–20 mC for 4 days. Disease symptoms (blighting of leaflets and sporulation) were evident by this time. Plants were transported to a greenhouse and kept on greenhouse benches for another 3 days. Seven days after inoculation, plants were transported to the laboratory and uninfected leaflets of third leaves were used for chemical assays and bioassays. Three trials of this experiment were conducted Assays for plant susceptibility to insects and pathogens H. zea bioassays. H. zea bioassays made use of a single leaflet from the leaflet pair adjacent to the terminal leaf. At least 8 leaflets from each treatment group (control, H. zea- , Pst-, or P. infestans-damaged, and when applicable, BTH-treated) were excised with a razor blade and placed on moist filter paper in Petri dishes. One third-instar H. zea larva was weighed and placed in each dish, the dishes were covered and sealed, and the larvae were allowed to feed for 24 h. The larvae used had all molted within 12 h of each other and were starved for 2–3 h before placement in dishes. After the 24-h feeding period, insects were starved for 2–3 h to void their guts and then weighed to the nearest 0n1 mg on a microgram scale (Cahn, Inc., USA). Relative growth rates were calculated using the formula of Waldbauer [47]. One of the experiments which tested the effects of P. infestans on suitability of leaves for noctuids used the beet armyworm, S. exigua, and not H. zea. Responses of H. zea and S. exigua to secondary chemicals are similar, although S. exigua is somewhat more sensitive to some of the secondary chemicals found in tomato. Methods used for S. exigua growth assay were identical to those used for H. zea growth assays. Bacterial speck disease assays. To assess the suitability of leaves for Pst, three leaflets on third leaves of plants were inoculated : a single leaflet of the pair next to the terminal leaflets, and the next pair of leaflets toward the stem. Plants were inoculated by gently rubbing entire upper surfaces of test leaflets with a cotton-tipped applicator saturated with the bacterial suspension (2i10( cfu ml−"). Plants were inoculated in a greenhouse during the evening. Plants were maintained in the greenhouse and symptoms were allowed to develop for 6–7 days ; number of lesions on each inoculated leaflet were counted at this time. Spectrophotometric assays Spectrophotometric assays of the activities of polyphenol oxidase and peroxidase were performed using an extract of a leaflet adjacent to the terminal leaflet of third leaves. Extracts were prepared using a tissue grinder to homogenize individual, weighed leaflets in 1n25 ml of pH 7 phosphate buffer (0n1 ) containing 7 % (w\v) polyvinylpolypyrrolidone. A volume (0n4 ml) of a 10 % solution of Triton X-100 was added with mixing to the homogenate and the homogenate was centrifuged at 6000 g for 15 min. The resulting supernatant was used directly as an enzyme source. Assays for polyphenol oxidase and peroxidase both measured the rate of formation of melanin-like material from phenolic substrates [42]. For polyphenol oxidase assays, 10 to 30 µl of enzyme extract were added to 1 ml of 2n92 m caffeic acid in pH 8

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potassium phosphate buffer (0n1 ) and the change in absorbance of the mixture at 470 nm was measured for 30 seconds. The procedure for assaying peroxidase activities was identical, but the substrate for peroxidase activities consisted of 5 m guaiacol with 0n02 m H O added as a cofactor. Polyphenol oxidase and peroxidase activities are # # reported as ∆OD min−" gm fresh weight−". RNA gel blot analyses Methods for assays of PINII, P4, and cyclophilin mRNA abundance in tomato leaves are described in a companion paper [18]. Replication and statistics Each potential interaction (e.g., the effect of infection by Pst on resistance to subsequent infection by Pst) was tested in at least two trials. There were generally between 12–16 plants in each treatment group in each trial, but limitations in amounts of leaflet tissue made it impossible to perform all chemical and biological assays using leaflets from a single plant. Each potential interaction was tested using no fewer than 8 plants per treatment per trial. Thus, data analyzed for a given interaction came from at least 16 plants from two trials. Likewise, data from at least 16 plants were analyzed for treatment effects on enzyme activities. As noted above, all possible chemical assays and biological assays were not always conducted in a single trial. However, the results from the different trials of each experiment are directly comparable. Methods used to damage plants, and the location and severity of damage, were consistent among trials of an experiment. Methods used to assay resistance were uniform throughout all experiments. Plant age and conditions for plant growth were also constant throughout the experiments. Data for each potential interaction or chemical effect were analyzed separately by ANOVA, with trial used as a blocking factor. Treatment by trial interactions are reported only when significant. Planned contrasts were used for comparisons of treatment means. The SAS statistical package (SAS Institute, Cary, NC, USA) was used for all analyses.

RESULTS

Plant-mediated interactions between Pst and H. zea Reciprocal, plant-mediated interactions in tomato foliage were investigated using Pst, the causal agent of bacterial speck in tomato, and the corn earworm, HelicoŠerpa zea, a common noctuid insect pest of tomato. The general scheme for these and subsequent experiments is outlined in Fig. 1. Terminal leaflets of tomato leaves were challenged by inoculating the leaflet with Pst or by allowing H. zea larvae to feed on the leaflet, and the susceptibilities of other leaflets on the same leaf to larvae and bacteria were compared with the susceptibilities of leaflets on undamaged plants. A third group of plants in each experiment was treated with BTH, a synthetic inducer of resistance to pathogens [20], and the treated leaflets subsequently evaluated for susceptibility to H. zea and Pst.

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Challenge treatment

Inducing treatment

Inducing treatment 48 h (insect) 120 h (BTH or pathogen)

Evaluation Challenge treatment

24 h (insect) 120 h (pathogen)

Gene expression and chemistry F. 1. Experimental system for induction and assessment of induced resistance and systemic acquired resistance in tomato leaves. Chemical and gene expression analyses were performed on leaflets of plants induced to express IR or SAR but not subsequently challenged with the biotic agents. In each experiment, these leaflets were collected at the same time when leaflets of a parallel set of similarly treated plants were challenged with biotic agents for subsequent resistance assessment. Leaflets on third leaves also were analyzed for oxidative enzymes and PINII and P4 mRNA at the indicated intervals unless otherwise noted.

Infection of terminal leaflets of third leaves of tomato plants by Pst resulted in a dramatic increase in the resistance of uninfected leaflets of the same leaf to subsequent infection by Pst. As shown in Table 1, leaflets from previously-inoculated leaves had between 35 % to 75 % fewer lesions than leaflets from previously-uninoculated leaves. This reduction in lesion number caused by prior inoculation was consistently greater than the reduction caused by application of BTH. Infection by Pst of the terminal leaflet of third leaves also reduced the suitability of uninfected leaflets of the same leaf for H. zea growth. As shown in Table 2, H. zea growth rates on leaflets from inoculated plants were 50 % to 80 % lower than on control plants. Leaflets to which BTH had been applied five days earlier supported higher rates of growth of H. zea than untreated leaflets. Feeding by larvae of H. zea on terminal leaflets of third leaves increased the resistance of undamaged leaflets of third leaves to subsequent infection by Pst. Table 3 summarizes results from 4 trials testing the effect of feeding damage on susceptibility to Pst. Leaflets from damaged plants showed, on average, 30 % fewer lesions than leaflets from undamaged plants. The increase in resistance to Pst caused by feeding of H. zea was generally not as pronounced as the increased resistance observed following application of BTH (36 % fewer lesions on average than controls).

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M. J. Stout et al. T 1 SeŠerity of bacterial speck disease caused by P. syringae pŠ. tomato (Pst ; number of lesions\leafletpSE) on leaflets from undamaged leaŠes, from leaŠes preŠiously inoculated with Pst, and from leaŠes treated with BTH No. of lesions\leaflet Trial No.

Control

Pst

BTH

1 21n3p3n6 11n4p2n0 16n6p2n7 2 26n2p3n0 17n6p2n4 18n4p2n4 3 7n9p1n4 1n8p0n3 4n3p0n6 ANOVA for effects of prior Pst infection and BTH treatment on severity of disease : Source of variation or contrast d.f MS F-ratio P treatment trial control vs. BTH control vs. Pst

2 2 1 1

536n78 2455n91 444n27 1040n93

10n86 49n70 8n99 21n07

0n001 0n001 0n004 0n001

T 2 RelatiŠe growth rates (gm gm−" day−"pSE) of H. zea larŠae reared for 24 h on leaflets from undamaged leaŠes, from leaŠes inoculated with P. syringae pŠ. tomato (Pst), and from leaŠes sprayed with BTH Relative growth rate of H. zea (gm gm−" day−"p) Trial No.

Control

Pst

BTH

1 0n46p0n10 0n23p0n05 0n76p0n11 2 0n52p0n03 0n11p0n03 0n75p0n02 ANOVA for effects of prior Pst infection and BTH treatment on suitability of leaflets for H. zea growth Source of variation or contrast d.f. MS F-ratio P treatment trial control vs. BTH control vs. Pst

2 1 1 1

1n37 0n01 0n56 0n82

39n10 0n27 16n08 23n24

0n001 0n61 0n001 0n001

Localized feeding by H. zea on the terminal leaflet of tomato leaves also induced resistance against H. zea in the undamaged leaflets of the same leaf. As shown in Table 4, growth rates of H. zea on leaflets from damaged plants were reduced 25 % to 65 % compared with growth rates on undamaged leaves. In contrast to results obtained from

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T 3 SeŠerity of bacterial speck disease caused by P. syringae pŠ. tomato (number of lesions\leafletpSE) on leaflets from undamaged leaŠes, from leaŠes damaged with H. zea, and from leaŠes treated with BTH No. of lesions\leaflet Trial No. 1 2 3 4 ANOVA for effects of Source of variation or contrast treatment trial control vs. BTH control vs. H. zea

Control 31n5p9n8 19n4p2n2 21n8p2n6 21n3p2n5 prior damage

H. zea

BTH

26n5p3n1 21n5p4n0 16n2p2n9 16n1p2n2 12n7p1n9 11n2p1n9 10n8p1n6 11n6p2n5 and BTH treatment on severity of disease

d.f

MS

F-ratio

2 3 1 1

862n46 806n61 1502n72 1034n47

8n0 7n48 13n94 40n16

P 0n001 0n001 0n001 0n002

T 4 RelatiŠe growth rates (gm gm−" day−"pSE) of H. zea larŠae reared for 24 h on leaflets from undamaged leaŠes, from leaŠes preŠiously damaged with H. zea, and from leaŠes sprayed with BTH Relative growth rate of H. zea (gm gm−" day−"pSE) Trial No. 1 2 3 ANOVA for effects of growth : Source of variation or contrast treatment trial control vs. BTH control vs. H. zea

Control 0n62p0n08 0n60p0n02 0n53p0n03 prior damage

H. zea

BTH

0n20p0n06 0n57p0n08 0n45p0n05 0n71p0n04 0n28p0n05 0n55p0n05 and BTH treatment on suitability of leaflets for H. zea

d.f.

MS

F-ratio

2 2 1 1

0n67 0n14 0n008 0n91

29n46 6n24 0n34 40n16

P 0n001 0n003 0n56 0n001

the experiments in which Pst was evaluated as the damaging agent, application of BTH had no statistically significant effect on suitability of foliage for H. zea in the three trials of this experiment, although growth rates were numerically higher on BTH-treated plants than on controls in two of the three trials.

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Relative enzyme activity

14 12

4

2

0

Pst

H. zea P. infestans Treatment

BTH

F. 2. Induction of polyphenol oxidase () and peroxidase ( ) activities following inoculation with P. syringae pv. tomato or P. infestans, feeding by H. zea larvae, or treatment with benzothiadiazole (BTH). Enzyme activities are expressed relative to activities in undamaged\ untreated plants (broken line). Statistical analyses are presented in Table 5.

Infection by P. infestans does not influence performance of noctuid herbiŠores Localized infection of leaves by P. infestans can induce systemic changes in plants resulting in SAR [46]. To test the hypothesis that an increase in resistance to noctuids is a general response to infection by pathogens in tomato foliage, the effects of localized infection by P. infestans on resistance of uninfected leaflets to noctuids were assessed. There were no differences between growth rates of either the beet armyworm S. exigua (trial 1) or H. zea (trials 2 and 3) on leaflets from infected plants and on leaflets from uninfected plants (data not shown). Induction of biochemical responses by Pst, P. infestans, H. zea, and BTH Biochemical analyses were conducted in conjunction with biological assays to assess the relationship between the treatment effects on pest resistance and the expression of hallmark activities that are putative resistance mechanisms. In all experiments, undamaged or uninfected leaflets from third leaves of four-leaf plants were used for these assays. As shown in Figs 2 and 3 and Table 5, infection of terminal leaflets by Pst resulted in the leaf-systemic induction of all chemicals assayed. The induction of polyphenol oxidase activity was greater than 10-fold ; the induction of peroxidase was weaker than this and inconsistent (i.e., a significant treatment by trial interaction was found for the induction of peroxidase by Pst, Table 5). Figure 3 shows that localized infection by Pst also strongly induced leaf-systemic expression of P4 and PINII mRNA. In some plants, P4 mRNA was detected in controls. However, pathogens and BTH always induced levels of P4 mRNA that greatly exceeded control levels. Feeding by H. zea on terminal leaflets caused a leaf-systemic induction of polyphenol oxidase activity but did not affect peroxidase activity (Table 5). The induction of polyphenol oxidase activity by H. zea feeding was not as marked as the induction by Pst infection (Fig. 2). Feeding by H. zea also induced the accumulation of proteinase inhibitor mRNA in undamaged leaflets of damaged leaves (Fig. 3). Levels of P4 mRNA were not affected by H. zea feeding (Fig. 3).

H. zea

PINII

PINII

P4

P4

P4

Cyclophilin

Cyclophilin

Cyclophilin

P. infestans

(C)

Control

(B)

125

Control

Control

(A)

P.s. pv tomato

Systemic defense-related signal interactions

F. 3. Representative gel blot analyses of systemic expression of PINII and P4 mRNA in subterminal leaflets following pathogen inoculation or insect challenge of the terminal leaflet on the third leaf of tomato plants. (A) Gene expression 5 days after inoculation with P. syringae pv. tomato. (B) Gene expression 2 days after challenge with larvae of H. zea. (C) Gene expression 5 days after inoculation with P. infestans. Expression of PINII varied from little to no expression in the inoculated leaves. Cyclophilin mRNA was measured as an internal standard to assess RNA loading.

Analysis of Šariance for the effects of four treatments on the actiŠities of the oxidatiŠe enzymes polyphenol oxidase and peroxidase. Treatment by trial interactions are shown only when significant Polyphenol oxidase Source of variation Pst infection treatment trial treatitrial H. zea feeding treatment trial P. infestans infection treatment trial BTH application treatment trial treatitrial *, P

0n05 ; ***, P

Peroxidase

d.f.

MS

F-ratio

d.f.

MS

F-ratio

1 2 2

26261n7 118n3 55n4

210n0*** 0n95 0n44

1 2 2

1688n2 670n3 610n7

27n1*** 10n7*** 9n79***

1 1

4665n8 1n5

1 1

499n5 189n2

1 1

17n6 3n0

1n0 0n2

1 1

42631n3 340n5

1 2 2

43n2 62n8 73n9

1n4 2n0 2n4

1 2 2

15979n0 534n6 457n0

22n05*** 0n01

3n18 1n21 21n57*** 0n17 113n3*** 3n79* 3n24*

0n001.

Infection of terminal leaflets by P. infestans and treatment of leaflets with BTH had qualitatively similar effects on plant chemistry (Table 5). Both treatments resulted in the induction of peroxidase activities, although the induction of peroxidase by BTH was weaker and less consistent than the induction of peroxidase by P. infestans (Table 5, Fig. 2). Both treatments strongly induced the expression of P4 mRNA well above

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levels found in controls (Fig. 3). Neither treatment induced polyphenol oxidase activity or PINII gene expression. DISCUSSION

The experiments reported here were initiated to investigate the coordination and integration of induced responses to pathogens and insects in tomato foliage. In particular, we investigated the potential for reciprocal, plant-mediated interactions between pathogens and insects and, to a lesser extent, the potential for increased vulnerability to insect herbivory in plants in which salicylate-regulated responses had been activated (both possibilities had been suggested by previous data ; 18, 32, 44). Our experiments provide strong evidence for reciprocal induced resistance involving certain pathogens and some arthropod herbivores of tomato foliage : localized infection by Pst induced systemic resistance to subsequent infection by Pst and to H. zea ; feeding by H. zea induced systemic resistance to H. zea and to Pst (Tables 1–4). This reciprocal induction of resistance cannot be attributed to direct interactions between the pathogen and insect, since initial infection or damage was spatially and temporally separated from the inoculations and feeding assays used to measure resistance. Nor was the induction of resistance against noctuids a generalized response to pathogen infection, since infection by P. infestans had no effect on the suitability of uninfected leaflets on infected leaves for H. zea or S. exigua growth. Note also that Pst is capable of strongly inducing both pathways, whereas P. infestans neither locally nor systemically induces proteinase inhibitors or responses typically associated with induced resistance to insects. Prior investigations of plant-mediated interactions between plant pathogens and arthropod herbivores have yielded inconsistent results [23]. Reproduction of the aphid, M. persicae, was reduced on tobacco leaves previously inoculated with tobacco mosaic virus [30], but, in a similar experiment [2], reproduction of the aphid Myzus nicotianae on tobacco leaves was unaffected by prior inoculation with tobacco mosaic virus. In several experiments performed by Potter and colleagues [1, 5], prior inoculation of cucumber plants with pathogens failed to affect the performance of a number of species of arthropods, although prior inoculation did induce systemic resistance against pathogens. Karban et al. [28] found that damage to cotton cotyledons caused by feeding of the mite, Tetranychus urticae, reduced subsequent susceptibility of the plants to Verticillium dahliae, and that plants previously inoculated with V. dahliae were poorer hosts for T. urticae. One possible explanation for the inconsistency in prior tests of plantmediated herbivore-pathogen interactions has been a failure to account for differences in responses of plants to different types of insect feeding and pathogen infection. The biological interactions observed in our experiments are explicable in terms of the biochemical changes observed in parallel. Considerable evidence implicates both polyphenol oxidase and serine proteinase inhibitors in the inducible resistance of tomato foliage to Lepidopteran larvae [16, 17, 26, 36, 42]. Thus, although it had not been previously demonstrated, the induction of resistance to H. zea by infection by Pst was expected on the basis of prior evidence showing the induction of proteinase inhibitors and polyphenol oxidase by Pst [18, 32 ; unpublished data]. The induction of resistance to H. zea by prior feeding by H. zea was expected both on the basis of the

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chemical responses induced by H. zea feeding and on the basis of previous demonstrations of resistance induced by this insect [40, 42]. PR proteins are thought to contribute to resistance to a variety of pathogens in tomato and other plants [8, 10, 35]. Indeed, disease symptoms caused by inoculation of leaflets with Pst were less severe on BTH-treated leaves, in which PR proteins but not octadecanoid-related responses were induced, than on untreated leaflets. The reduced severity of bacterial speck disease symptoms on leaflets from H. zea-damaged leaves (see also ref. 44) suggests that octadecanoid-related responses could also contribute to protection against Pst, although the resistance induced by H. zea was on the whole weaker than the resistance induced by treatment with BTH. The strongest resistance against Pst was induced by prior inoculation with Pst, which induced both octadecanoidrelated proteins and PR proteins. The resistance induced by Pst against Pst may be rendered by the combinatorial action of products induced by multiple signals in concert with jasmonate and salicylate, and that may offset salicylate’s suppression of jasmonate action. The failure of chemical and biological inducers of PR proteins (BTH and P. infestans, respectively) to induce resistance to H. zea and S. exigua indicates that these proteins are not sufficient for induced resistance against noctuids in tomato under our experimental conditions. In fact, induction of salicylate-regulated responses may actually increase the suitability of leaflets for H. zea : growth rates of H. zea on BTH-sprayed leaflets were higher than growth rates on control leaflets in one set of experiments (Table 2), and significant elevation of growth rates of H. zea and other insects on BTH-treated leaves has been observed in subsequent research in both greenhouse and field-grown tomato plants (unpublished data). This increase in suitability of BTH-treated leaves to noctuids may have resulted partially from inhibition by BTH of induced responses to damage. However, signaling interference probably does not fully explain the results observed in our experiments. Feeding assays were of short duration (24 h) and, because there is a 12- to 24-h lag between damage and expression of induced proteins in tomato [43], inhibition of induced responses could have accounted for differences in leaflet suitability during the latter portions of feeding assays only. Thus, it may be that the induction of PR proteins or other salicylate-related responses improved the suitability of foliage for H. zea in a more direct fashion. This evidence, of course, does not negate the possibility that PR proteins may contribute to resistance against other insects. Broadway et al. [7] recently demonstrated decreases in insect growth on artificial diets supplemented with chitinases and glucanases, two classes of PR proteins associated with induced plant resistance to pathogens [10, 25]. Peroxidases have been implicated as a contributing factor in resistance against both pathogens and insects [4, 12, 22]. The induction of peroxidase activity in our experiments was consistently associated with the induction of P4 mRNA and the induction of resistance against Pst. However, the consistent failure of treatments that induce peroxidase to induce resistance against H. zea in tomato—inoculation with P. infestans and treatment with BTH (this paper), ultraviolet light [45], and feeding by aphids [44] - argue against a role for peroxidases in induced resistance against H. zea in tomato. On the other hand, overexpression of peroxidase in transgenic tobacco foliage is reported to confer some resistance against H. zea [12]. The differences between our results and those of Dowd and Lagrimini [12] may be due to differences in host

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plant, differences in the methods by which expression of peroxidase was increased, or differences in the expression and relative importance of specific peroxidase isoforms in the response [39]. Tomato has been an historically important model for the investigation of inducible responses to both pathogens and insects. Our results, considered together with this prior work, permit inferences about the coordination and integration of the induced defenses of tomato, and perhaps of other plants as well, against diverse pests. The foliage of tomato contains a variety of secondary chemicals, defense-related proteins, and structural elements thought to be involved in induced resistance against pathogens and insects [13, 36]. This array of defensive factors is divided into coordinately-inducible, integrated sets, with each set containing multiple secondary compounds and defenserelated proteins. Moreover, these coordinately-inducible sets are differentially inducible, such that different sets are activated by different pathogens and insects, but these sets cannot be strictly dichotomized as insect-inducible or pathogen-inducible. Induced defense against a given insect or pathogen appears to be attributable to the combined action of the multiple components within an inducible set. A given set may be involved in defense against both pathogens and insects ; the octadecanoid pathway, for example, contributes to induced resistance in tomato foliage against H. zea and possibly P. infestans [9]. In some cases, defense against a pest may be rendered by the combined action of more than one set, as was probably the case with the induction of resistance to Pst by prior inoculation with Pst. Importantly, the expression of some sets may be inhibited by the expression of others (e.g., the inhibition of the octadecanoid pathway by salicylic acid ; 11, 37, 38), with a corresponding detrimental impact on host resistance against the particular pests sensitive to the inhibited set(s). There are indications in tomato [42] and other plants [37, 38] for defense response pathways in addition to the octadecanoid and salicylic acid pathways. Studies such as ours, in which induced resistance to pathogens and insects is studied in parallel with chemical responses, are needed to clarify the above inferences and examine them in other plant\pest systems. Important in such studies will be the use of defined mutants such as salicylate- and jasmonate-modified plants [19, 24]. Our results affirm the importance of the inclusion of a battery of pests when evaluating the effects of novel chemical and biological agents that operate through induced plant resistance to fully assess their potential for integrated disease and insect control. We thank Charles S. Gasser, Charles A. Ryan, and Jan A. L. van Kan for plasmids containing sequences for use as hybridization probes, and Michael J. Coffey and Donald C. Cooksey for pathogen isolates. This work was supported in part by USDANRI Grants No. 91–37303–6504 and No. 96–35303–3238 awarded to R. M. B., No. 95–37302–1802 awarded to S. S. D., M. J. S., and R. M. B. and by a Jastro-Shields Research Scholarship and UCD Humanities Research Award awarded to A. L. F. A. L. F. received support from Conselho Nacional de Desenvolvimento Cientı! fico Tecnolo! gico (CNPq), Brazil. REFERENCES 1. Ajlan AM, Potter DA. 1991. Does immunization of cucumber against anthracnose by Colletotrichum lagenarium affect host suitability for arthropods ? Entomologia Experimentalis et Applicata 58 : 83–91.

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