Signal interactions in pathogen and insect attack: expression of lipoxygenase, proteinase inhibitor II, and pathogenesis-related protein P4 in the tomato,Lycopersicon esculentum

Physiological and Molecular Plant Pathology (1999) 54, 97–114 Article No. pmpp.1998.0192, available online at http :\\www.idealibrary.com on

Signal interactions in pathogen and insect attack : expression of lipoxygenase, proteinase inhibitor II, and pathogenesis-related protein P4 in the tomato, Lycopersicon esculentum A. L. F"*, M. J. S#†, J. S. T#, S. S. D#‡ and R. M. B"§ " Department of Plant Pathology and # Department of Entomology, UniŠersity of California, DaŠis, CA 95616, USA (Accepted for publication NoŠember 1998)

Pathogens and insects can elicit different sets of plant host responses, supporting the hypothesis for control by different signaling pathways. To evaluate the potential for signal interaction in plants attacked by pathogens and insects, the mRNA abundance for lipoxygenase (LOX), a woundinducible proteinase inhibitor (PINII), and a pathogenesis-related protein (P4) was evaluated in tomato leaves following challenge with a variety of agents. PINII and P4 expression was determined as these proteins are induced in tomato leaves characteristically following attack by certain insects or pathogens, respectively. Expression studies of LOX, PINII, and P4 indicate that their induction in tomato does not follow a strict pattern based on the type of biologic inducer (insect vs. pathogen) or chemical treatment, with each specific treatment inducing a distinct pattern of gene expression. However, plants induced to express disease resistance with the synthetic salicylate mimic benzothiadiazole-7-carbothioic acid S-methyl ester were compromised in their expression of the wound- or jasmonate-activated PINII, consistent with an observed increase in susceptibility to insect herbivory reported in a companion study. The results do not support the hypothesis for a strict dichotomy of signaling by insects and pathogens of LOX, PINII and P4 in tomato, but point to a potential vulnerability of acquired resistance evident at the levels of gene expression and response to insect attack. # 1999 Academic Press

INTRODUCTION

In higher plants, the consequences of wounding from insect herbivory and infection by pathogens can be different, with differences manifested at the levels of gene expression, induced chemistry, and host resistance to further challenge [8, 21, 52, 54 ]. However, * Present address : Section of Plant Biology, University of California, Davis, CA, 95616. † Present address : Department of Entomology, Lousiana State University, Baton Rouge, LA, 70803, USA. ‡ Deceased. § To whom correspondence should be addressed. Abbreviations used in text : AA, arachidonic acid ; BTH, benzothiadiazole-7-carbothioic acid S-methyl ester ; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A ; JA, jasmonic acid ; LOX, lipoxygenase ; P4, pathogenesis-related protein 4 ; PIN, proteinase inhibitor ; PR, pathogenesis related ; Pst, Pseudomonas syringae pv. tomato ; SA, salicylic acid ; SAR, systemic acquired resistance. 0885–5765\99\030097j18 $30.00\0

# 1999 Academic Press

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Pathogen signals

Membrane lipids

ROS, lesions, HR, apoptosis

Linolenic acid lipoxygenase

?

Fatty acid peroxides ?

Salicylate

Jasmonates

Wound responses • proteinase inhibitors • polyphenol oxidase • steriod glycoalkaloids • induced resistance to insects and some pathogens

Infection responses • PR-proteins • phytoalexins • wound response suppression • systemic acquired resistance

F. 1. Model for generation of signals to engage different local and systemic stress responses following pathogen and insect attack in the Solanaceae. ROS, reactive oxygen species ; HR, hypersensitive response. See text for explanation.

insect attack and pathogen infection can both trigger oxidative reactions in the plant that contribute to induced responses and symptoms. Polyphenol oxidases and peroxidases are induced, with concomitant phenolic oxidation [53 ], reactive oxygen species (ROS) are generated [3–5 ], and lipoxygenases (LOX) participate in the peroxidation of membrane lipids and synthesis of signaling molecules [31, 51 ]. Although it is tempting to consolidate signaling in the plant following challenge by multiple pests within a common rubric, particularly within the context of defense and resistance, the literature is replete with evidence to suggest that such a grouping is simplistic [8, 34, 52 ]. The signaling pathways controlling responses to insects vs. those to pathogens and their toxins or elicitors lead to different products, and differ with respect to their impact on local and systemic resistance to insects or pathogens. As shown in the model depicted in Fig. 1, damage from certain types of insect herbivory is transduced in part through the octadecanoid pathway, involving the generation of oxylipins from the peroxidation of endogenous polyunsaturated fatty acids such as linoleic and linolenic acids [31 ]. In solanaceous species, the jasmonates, oxylipins derived from linolenic acid, can elicit responses that are characteristically associated with wounding [10 ] or damage by chewing insects, such as proteinase inhibitor accumulation, induction of a specific isoform of HMG-CoA reductase, and steroid glycoalkaloid synthesis [11, 12, 15, 24, 61 ]. In contrast, pathogens and their metabolites, such as the fungal elicitor arachidonic acid (AA) and LOX metabolites of AA, can trigger the accumulation of PR-proteins, isoforms of HMG-CoA reductase different from those induced by jasmonate, sesquiterpene phytoalexins, and pro-

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grammed cell death or apoptosis [7, 12, 16, 60 ]. These results indicate that a dramatic impact on cellular metabolism can be achieved through the action of specific lipidderived signals, and suggest the participation of LOX-mediated factors in this signaling. Although initial models depicted jasmonates as having a general signaling role for defense responses against both insects and pathogens [24 ], other findings have prompted a reevaluation of this hypothesis. For example, salicylic acid, produced in plants following challenge by a variety of pathogens, is generated locally and systemically during necrotic lesion formation and induces PR-proteins [19 ]. Salicylate and its synthetic mimics do not induce necrosis or phytoalexin accumulation, but condition systemic acquired resistance (SAR), and may interfere with jasmonateregulated responses [17, 18, 42 ]. The extent to which the signals associated with these responses are synergistic or counteractive is unresolved, and scattered reports suggest some convergence in signaling. For example, a study by Xu et al. [62 ] illustrates the potential for synergy between specific combinations of signals in their elicitation of certain PR-proteins. Ethylene may be synergistic with jasmonate in its induction of osmotin, and jasmonate enhances SA induction of PR-1. Jasmonate at high concentrations is reported to induce resistance to the late blight fungus Phytophthora infestans (Mont.) de Bary [14 ], similar to its ability to induce resistance to certain insects [57 ], and induces proteins in plants that may have antimicrobial action [43 ]. Pseudomonas syringae pv. tomato (Okabe) Young, Dye and Wilkie (Pst) induces systemic accumulation of proteinase inhibitors in tomato, but the spatial and temporal expression is somewhat different from that observed following artificial injury [41 ]. Recent evidence with jasmonate perception and biosynthesis mutants of Arabidopsis clearly implicate a role for jasmonates in defense against the opportunistic pathogen Pythium irregulare [59 ] Of concern in crop protection is the potential for the short-circuiting of pathways that lead to induced resistance to insects or to SAR against certain pathogens. Is there a detrimental impact on resistance of the plant to insect challenge if pathogen response pathways are triggered, and vice versa ? The recent availability of new chemicals for use in agriculture that trigger SAR makes resolution of these issues especially critical [36 ]. In a related study, we provide evidence that, indeed, engagement of SAR in tomato can result in increased susceptibility to herbivory by insects [56 ]. In this study, different treatments were imposed on tomato leaves and their effects assessed on the mRNA abundance in the challenged leaflets for LOX, a proteinase inhibitor (PINII), and the tomato pathogenesis-related protein P4. Our objectives were to establish in one system under uniform experimental conditions patterns of gene expression that reflect the degree to which the salicylate and jasmonate pathways are engaged locally following treatment of leaves with various agents. The agents used were : arachidonic acid (AA) ; a synthetic inducer of SAR, benzothiadiazole-7carbothioic acid S-methyl ester (BTH) ; jasmonic acid (JA) ; the late blight fungus, Phytophthora infestans ; the bacterial speck pathogen Pseudomonas syringae pv tomato (Pst), larvae of the corn earworm, HelicoŠerpa zea (Boddie) ; and the aphids Macrosiphum euphorbiae (Thomas) and Myzus persicae (Sulzer). LOX expression was assessed in these different contexts because of its potential for participating in fatty acid signaling in plants challenged by either insects or pathogens and for generating highly reactive and

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membrane disruptive lipid peroxides. PINII and P4 were measured because they provide markers for injury associated with certain insects and pathogens, respectively [30,55,58 ]. In addition, the interaction of several of these signals on gene expression was investigated. The gene expression studies reported here were prerequisite to, and provided the framework for, a companion study of signal interaction in systemic induced resistance in tomato to insects and pathogens [56 ]. MATERIALS AND METHODS

Plants and growth conditions Tomato (Lycopersicon esculentum Mill. cv. New Yorker) plants were grown in a greenhouse with supplemental lighting provided by a 1000 Watt sodium vapor lamp on a 16 : 8 light : dark cycle. In most experiments, plants in the four-leaf stage were used, and the treatments were imposed on the entire third leaf. Only the three apical leaflets of the leaf were collected for analyses. At specific time points leaflets were excised from two to three plants, pooled, frozen immediately in liquid nitrogen and stored at k80 mC until extraction. In some experiments with jasmonic and arachidonic acids, leaflets of similar size and maturity as those from four-leaf stage plants were excised from mature plants, placed in vials containing water, treated and incubated in humid plastic containers. Experiments were conducted either in the greenhouse or in a growth chamber at 25 mC, both with a 16 hr photoperiod. In the growth chamber, plants were illuminated from 06n00 h to 20n00 h with a combination of cool white fluorescent and incandescent lamps giving an intensity of approximately 112 W m−# irradiance at the leaf canopy during the photoperiod. For experiments with P. infestans, leaflets were detached at the time of inoculation and placed in petri dishes within a growth chamber at 20 mC to permit disease development. Pathogenic agents Sporangia for inoculations were prepared from P. infestans race 1 maintained on rye seed agar as described in previous publications [7 ]. The isolate was provided by M. Coffey, University of California, Riverside. Inoculation was accomplished by gently applying a spore suspension containing 10& sporangia ml−" with a small paint brush to the upper surface of the leaves. Controls consisted of plants treated with water. P. syringae pv. tomato, strain Pst23, was a gift of D. Cooksey, Department of Plant Pathology, University of California, Riverside. For each experiment, Pst was obtained from a frozen bacterial stock and grown on King’s B agar medium for 48 hr at 28 mC. The bacteria were harvested and diluted in water to a concentration of 10( cfu ml−", as indicated. The bacterial suspension was applied gently to the upper surface of the leaflets with a cotton swab, and the plants placed in the greenhouse until evaluation. Leaflets were harvested at various times after inoculation and frozen as described above. Insects H. zea (corn earworm) larvae were maintained as described previously [52 ]. Fourleaf tomato plants were damaged with these larvae by placing a single fourth-instar larva on the third leaf of plants and enclosing the entire leaf in a polyester mesh sleeve.

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Polyester sleeves were closed at one end and had a Velcro2 closure at the other. Eight to twelve hours were usually sufficient for larvae to damage most or all of the leaflets of the third leaf. Damaged leaflets were harvested and frozen in liquid nitrogen 24 and 48 h after placing larvae on the plants. Control plants received sleeves but no larvae. A colony of potato aphids (M. euphorbiae) initially obtained from field-infested tomato plants was maintained on tomato plants in a greenhouse. At the time of the experiment, plants in the four-leaf stage were artificially infested on their three terminal-most leaflets of the third leaf with approximately 40 aphids as described previously [53 ]. Control plants were not manipulated in any way. Aphids were allowed to feed for a week, where they remained on the third leaf. After this, aphids were removed and the damaged leaf was harvested and frozen as described earlier. Although we had no indications that the M. euphorbiae colony contained viruses that might influence plant responses in our initial experiments, we eliminated this possibility by verifying our results with green peach aphids (M. persicae) that were known to be nonviruliferous. These aphids were reared on radish (Raphanus satiŠus L. cv. White Icicle) in a growth room providing a 16 h photoperiod and 23 mC [20 ] and were provided by B. Falk. These aphids were evaluated for comparison with M. euphorbiae. Chemicals and reagents Jasmonic acid (JA) was prepared from methyl jasmonate (a mixture containing 90n6 % 1R,2R and 8n1 % 1R,2S ; Bedoukian Research, Inc., Danbury, CT. USA) as described by Farmer et al. [22 ]. JA was sprayed on individual leaves to run-off with a chromatography sprayer. Controls were similarly treated with water. The elicitor arachidonic acid (AA ; Sigma, St. Louis, MO, USA) was freshly prepared before each use as previously described [6 ]. In some experiments, AA was prepared in acetone and diluted to a 25 % acetone in water suspension to enhance solubilization. In these experiments, control leaves were treated with a 25 % acetone solution. Suspensions of AA were sprayed on the upper surface of the leaves until runoff, as described above. Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH, CGA 245704, 50 WG ; ref. 47) was prepared in water at 1n2 m, a concentration reported to induce PR protein accumulation in tobacco [26 ], and was a gift from Novartis, Inc., Greensboro, NC, USA. RNA isolation and gel blot analyses Total RNA was prepared from tomato leaves as described previously [12 ]. RNA (20 µg per lane) was fractionated by electrophoresis through 1 % agarose gels containing formaldehyde and transferred to Nytran membranes. Hybridization of plox1 and PINII probes to RNA blots was routinely carried out in 50 % formamide, 5iSSC, 0n02  sodium phosphate buffer, 1iDenhardt solution, 0n1 % SDS, 0n01 % sodium pyrophosphate, 200 µg ml−" salmon sperm DNA, 42 mC, and washed initially at low stringency (2iSSC, room temperature) followed by a more stringent wash (0n1iSSC, 0n1 % SDS at 40 mC). The P4 probe was hybridized to gel blots and the blots washed using the conditions described by van Kan and colleagues [58 ]. The amount of radioactively labeled probe hybridized to each RNA sample was estimated with a twodimensional radioisotope imaging system (Ambis, Inc., San Diego, CA, USA), or by

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area scanning of digitized autoradiograms using SigmaScan software (Jandel Scientific, San Rafael, CA, USA). Equal loading of RNA samples was verified by hybridization of the blots with a tomato cyclophilin DNA probe and by staining of rRNAs with ethidium bromide. Cyclophilins are ubiquitous and abundant proteins involved in folding of many proteins, among other functions [28 ]. Treatment of plants with salicylic acid and stresses, such as wounding, may interfere with cyclophilin transcript accumulation [39, 40 ]. However, under the conditions used in this study, the intensity of the signals for cyclophilin obtained with the cyclophilin probe agreed with the intensity of the rRNAs obtained after staining. Therefore, the abundance of cyclophilin mRNA appeared to provide a satisfactory measure of RNA loading. Since LOX and cyclophilin probes could be used simultaneously, the hybridization and washing conditions were the same. All experiments were performed at least twice. Preparation of DNA probes A probe containing the entire open reading frame of the plox1 potato tuber cDNA was used for all the LOX analyses [25 ]. plox1 was amplified from its vector pBluescript II SK(j) (Stratagene, La Jolla, CA, USA) by PCR using primers specifically designed to amplify the entire open reading frame. A 480-bp fragment from the insert of a plasmid vector containing a tomato cyclophilin clone [27 ] was obtained by digestion of the vector with EcoRI. PINII [30 ] was obtained by digestion of the plasmid vector pT2–47 containing the tomato PINII with EcoRI and HindIII, which produced a fragment of about 700-bp. The insert from a plasmid vector containing a sequence for the tomato PR-protein P4 [58 ] was amplified by PCR using commercial primers, T3 and T7. The PCR and\or digestion products were separated in 0n7 % agarose (FMC, Inc.) in TAE buffer. The target fragments were excised from the gel using the QIAEX Gel Extraction Kit (QIAGEN, Chatsworth, CA, USA) according to the manufacturer’s directions. The DNA probes were radioactively ($#P-CTP) labeled with the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN, USA) following the manufacturer’s protocol. Gel blots containing RNA from treated and nontreated leaf samples were first hybridized with the plox1 and cyclophilin probes. After analysis, the blots were then stripped and hybridized with the pinII probe, and subsequently, stripped again and hybridized with the p4 probe. Hybridized blots were stripped by placing them three times, 15 min each time, in a boiling solution containing 2 m sodium phosphate buffer, 0n025 m EDTA, 0n005 % sodium pyrophosphate, and 0n05 % SDS (modified from ref. 2). Signal interaction studies Effect of BTH. BTH strongly induces PR-protein accumulation and SAR in tobacco at a concentration of 1n2 m [26 ], and a preliminary concentration-response study indicated that this was a concentration that maximally induced P4 mRNA in tomato (unpublished results). Therefore, this concentration was selected to test for its potential interaction with the signal molecules and treatments under investigation in this study. Experiments were conducted at least twice. Tomato plants in the four-leaf stage were sprayed with BTH and controls were sprayed with water. The treated plants were kept for 6 days in a growth chamber at 25 mC with artificial lighting as described above.

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Leaflets on the third leaf were then challenged with a second treatment or inoculated as described below, and evaluated and analyzed after several days, depending on the specific treatment. The effect of BTH pretreatment was evaluated on arachidonate action (13 m AA in 25 % acetone ; 25 % acetone control), on late blight disease (10& sporangia ml−" of P. infestans race 1), and on bacterial speck disease (10( cfu ml−" of Pst). With the pathogens, controls consisted of leaflets treated with water for the second treatment. For bacterial speck evaluations, lesions were counted on leaves six days after inoculation. The results reported are the average lesion number per leaf from 12 plants for each treatment per trial. For late blight evaluations, leaflets were excised from the plant, seven days after BTH treatment and placed in Petri dishes upon damp filter paper. The leaflets were inoculated with five 20 µl drops of the spore suspension. The Petri dishes were sealed with parafilm and placed in a plastic box in a growth chamber at 20 mC under the same light conditions described above, and evaluated four days later. At the end of the experiments, inoculated leaflets from three pretreated and three control plants were combined, extracted for RNA, and analyzed for the abundance of transcripts for LOX, PINII, and P4. Effect of BTH and JA. Four-leaf stage tomato plants grown in the greenhouse were simultaneously sprayed on the third leaf with 1n2 m BTH, 1 m JA, or with a mixture of BTH and JA at these concentrations prepared as described above. Plants were maintained in the greenhouse, and four days later an adjacent leaflet from the third leaf was collected, frozen in liquid N , and kept at k80 mC until required for RNA # extraction. RESULTS

Lipid signaling of LOX, PINII, and P4 expression Jasmonate-induced gene expression. Treatment of tomato leaves with JA has been reported to induce LOX and PIN transcript accumulation [23,32 ]. To verify this under our experimental conditions, we applied JA to tomato leaves and found that LOX and PINII transcripts were induced within 3 h after treatment, although LOX expression was transient and weak (data not shown). At the concentration used, JA did not induce any visible symptoms in the leaves over the course of the experiments, which lasted not more than 4 days. PINII transcripts accumulated to high levels, a result similar to that reported for the response of PINI mRNA by Heitz et al. [32 ]. Transcripts for the PRprotein P4, which is serologically similar to the tobacco PR-1 [58 ], a hallmark of SAR, were not induced in tomato plants following jasmonate treatment (data not shown). Arachidonate-induced gene expression. The elicitor action of arachidonate is also thought to involve LOX activity and arachidonate is metabolized by plant cells to several biologically active eicosanoids [9, 45 ]. To compare octadecanoid and eicosanoid signaling in tomato, leaflets were treated with AA at a concentration that induces a maximal response. As shown in Fig. 2, AA, a fungal elicitor capable of inducing SAR [13 ], induced LOX mRNA within 4 hr after treatment and strongly induced P4 mRNA, both of which remained high during the course of the experiment. In contrast to JA treatment, AA induction of PINII expression was inconsistent, but generally was not significantly different from the control (data not shown).

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1.0 (a) 0.8

0.6

0.4

AA

0.2

0

24

48

72 Time (h)

96

120

1.0 Relative P4 mRNA abundance

(b) 0.8

0.6

0.4 AA Control

0.2

0

24

48

(c)

72 Time (h)

96

120

Time (h) 0 0.5 1

2

4

6 12 18 24 36 48 72 93 120

LOX

P4

Cyclophilin F. 2. For legend see opposite.

Signal-response specificity to pathogens and insects 0

24

105 48

96

LOX

P4

Cyclophilin

F. 3. Effect of P. infestans infection on the accumulation of LOX and P4 mRNA in tomato leaves. Tomato leaves were inoculated with sporangia (10& ml−") of P. infestans race 1. RNA was extracted at various times (h) after treatment and analyzed for the abundance of transcripts for LOX, P4, and cyclophilin.

Pathogen induction of LOX, PINII, and P4 expression P. infestans and Pst elicit different gene expression patterns in tomato leaŠes. Two pathogens that cause diseases with different symptoms were compared for their ability to elicit LOX, PINII, and P4 gene expression. Macroscopic symptoms were apparent on tomato leaves within 48 to 72 hr after inoculation with sporangia of the late blight fungus P. infestans, with extensive colonization and sporulation by 96 to 120 hr. Control leaves were healthy and remained green and turgid for the duration of the experiments. As shown in Fig. 3, inoculated tomato leaves accumulated LOX mRNA simultaneously with the initiation of visible lesions, a pattern similar to that observed in inoculated potato foliage [25 ]. No LOX induction was observed in the controls. Inoculation with P. infestans did not significantly induce PINII mRNA accumulation in the inoculated leaflets. PINII transcripts were detected only after extensive symptom development on the leaf 96 hr after inoculation (data not shown). In contrast, P4 transcripts were F. 2. Effect of AA treatment on LOX and P4 mRNA accumulation in tomato leaves. (a) LOX transcript abundance. (b) P4 transcript abundance. (c) RNA gel blot hybridized with probes for LOX, P4 and cyclophilin. Tomato leaves were treated with AA or 25 % acetone (control). RNA was extracted at various times (h) after treatment and analyzed for the abundance of transcripts for LOX and cyclophilin. Values are the means and SE from three separate experiments and are expressed relative to the maximum level of transcript abundance detected with (a) plox1 or (b) P4 DNA probes. Levels of LOX mRNA in control leaflets were negligible. —4—, AA ; — —, Control.

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24

72

Pst 96

120

0

24

72

96

120

(h)

LOX

PINII

P4

Cyclophilin

F. 4. Effect of infection by Pst on the accumulation of LOX, PINII, and P4 mRNA in tomato leaves. Tomato leaves were inoculated with Pst (10( cfu ml−"). RNA was extracted at various times (h) after infection and gel blots were hybridized with DNA probes to detect LOX, PINII, P4, and cyclophilin mRNA.

strongly induced after P. infestans infection in tomato leaves (Fig. 3). No P4 transcripts were detected in the controls in these experiments. Tomato leaves inoculated with Pst (10( cfu ml−") induced typical bacterial speck lesions that were apparent by 72 to 96 h after inoculation. LOX transcripts could be detected by 24 hr after inoculation, and increased during the time course of observation. As shown in Fig. 4, PINII and P4 were strongly induced and could be detected within 24 hr following inoculation. Insect induction of LOX, PINII, and P4 Feeding by the caterpillar H. zea, a chewing insect, damaged leaves extensively such that within 48 to 72 h after challenge very little leaf area remained. As shown in Fig. 5A, caterpillar feeding induced a weak transient accumulation of LOX mRNA detectable within 24 h after challenge. By 48 h, LOX transcripts could no longer be detected. Injury to tomato leaves by the aphid M. euphorbiae, a sucking insect, induced LOX and P4 expression, whereas PINII was not induced by this agent (Fig. 5B). The same result was obtained in a similar experiment using the aphid M. persicae (data not shown). In contrast to aphid injury, H. zea larvae strongly induced PINII mRNA, but only weakly induced P4 expression, a signal detected only after 1-week exposure of the blots.

Signal-response specificity to pathogens and insects

LOX

LOX

PINII

PINII

P4

P4

Cyclophilin

Cyclophilin

Aphid

Control

(b) H. zea

Control

48 h H. zea

24 h Control

(a)

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F. 5. Effect of feeding by corn earworm larvae (H. zea) or aphids (M. euphorbiae) on the accumulation of LOX, PINII, and P4 mRNA in tomato leaves. (a) Tomato leaves damaged by H. zea larvae. (b) Tomato leaves were damaged by M. euphorbiae. RNA was extracted 24 and 48 h after the larvae were introduced or one week after aphid feeding and gel blots were hybridized with DNA probes for detection of LOX, PINII, P4 and cyclophilin mRNA.

Signal interaction studies BTH does not affect AA activity. Since both the synthetic salicylate mimic BTH and AA can induce SAR, they were compared for their ability to induce the genes of interest and to see if there was any synergy in their induction of leaf responses when applied in combination. AA induced necrotic areas in treated tomato leaves, with initial symptoms apparent within several hours after treatment. Treatment with BTH alone had no visible effect on leaves and appeared to give the same effect as the water-treated controls. To verify that BTH under our experimental conditions was physiologically active, tomato plants were sprayed with BTH or with water, and RNA was extracted from the leaves and analyzed for the presence of P4 transcripts. As shown in Fig. 6, BTH strongly induced P4 transcripts. Symptoms in detached tomato leaves pretreated with BTH and then treated with AA resulted in no visible effect on AA action. Cross interference of gene induction by BTH and JA. To determine if engaging SAR with BTH compromises the expression of the jasmonate-activated PINII, plants were sprayed with water (control), BTH, JA, or simultaneously with a combination of BTH and JA. BTH alone strongly induced P4 transcripts (Fig. 6). However, when BTH was simultaneously applied with JA, the levels of P4 transcripts decreased relative to the levels induced by BTH (0n71p0n14 relative to BTH alone). The combination of BTH and JA also diminished PINII transcript accumulation relative to the PINII levels induced after JA treatment (0n76 relative to JA alone ; Fig. 6).

JA/BTH

JA

BTH

A. L. Fidantsef et al. Control

108

PINII

P4

Cyclophilin

F. 6. Effect of application of BTH and JA on PINII and P4 mRNA accumulation in tomato leaves. Tomato plants at the four-leaf stage were sprayed with BTH (1n2 m), JA (1 m), or simultaneously sprayed with a mixture containing BTH (1n2 m) and JA (1 m). Four days later, an adjacent leaflet of the third leaf was removed for RNA extraction and RNA gel blots were hybridized with DNA probes for detection of PINII, P4 and cyclophilin mRNA.

BTH induces resistance to Pst but not to P. infestans. To determine if BTH induces resistance to the bacterial speck pathogen Pst, tomato plants were pretreated with BTH or water and inoculated by brushing bacteria onto the leaf surfaces. As shown in Fig. 7a, BTH induced P4 mRNA accumulation, suppressed the bacterially induced accumulation of PINII transcripts, and significantly reduced the number of bacterial speck lesions (Fig. 7b). BTH induces resistance in tobacco to Phytophthora parasitica [26 ] and in wheat to other fungal pathogens [29 ]. However, its impact on late blight disease caused by P. infestans has not been reported. To determine this, tomato leaves were treated and inoculated, and in two trials, late blight disease symptoms in inoculated leaves were as severe in the BTH-pretreated leaves as in the water-pretreated controls. There was no apparent protection against P. infestans afforded by BTH at a concentration that maximally induces P4 mRNA in tomato leaves and reduces the severity of bacterial speck disease.

PINII

P4

H2O/Pst

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(b) 50 45 Number of lesions per leaf

LOX

H2O

BTH

(a)

BTH/Pst

Signal-response specificity to pathogens and insects

40 35 30 25 20 15 10

Cyclophilin

5 0

Trial 1

Trial 2

F. 7. Effect of BTH pretreatment on gene expression and disease severity in tomato leaves inoculated with Pst. (a) RNA gel blot of LOX, PINII, and P4 mRNA accumulation. (b) Bacterial speck disease severity (lesion number). Tomato leaves were pretreated with BTH or water and then six days later inoculated with Pst (10( cfu ml−") or left noninoculated. (a) RNA was extracted six days after the second treatment and RNA gel blots were hybridized with DNA probes for detection of LOX, PINII, P4 and cyclophilin mRNA. (b) Values are the means and SE of the number of lesions per leaf using twelve plants per treatment for each experiment. Treatments are significantly different (P l 0n001) by t-test for both trials. , leaves pretreated with water ; O, leaves pretreated with BTH.

DISCUSSION

This study assessed the potential interaction between plant signaling pathways that are triggered following attack by certain pathogens and insects. Proteinase inhibitor induction and PR-protein gene expression were used as hallmark indicators of responses to insect injury and infection, respectively, to assess the degree of independence or overlap in signaling in tomato leaves following treatments. LOX expression at the levels of mRNA accumulation provided a measure of the extent to which this lipid peroxide generating pathway is engaged in the various contexts, since message accumulation precedes or temporally parallels induced LOX enzyme activity in potato [25 ] and tomato leaves (unpublished results). As summarized in Table 1, experiments in general, corroborated the association of PINII expression with wounding and feeding by noctuid insect larvae, and P4 expression with fungal and bacterial infection. However, these studies also revealed exceptions to this, with overlap in response pathways especially in the cases of aphid feeding, which induced P4 but not PINII expression, and of infection by Pst which induced both. LOX activation provided a somewhat less discriminating measure of the type of stress imposed on tomato leaves. The marked differences in responses to feeding by the insect species tested, and the similarities in responses to infection by P. infestans and feeding by aphids, illustrate the point that taxonomic identity is not a reliable predictor of the types of responses

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A. L. Fidantsef et al. T 1 Summary of the effects of different types of injury or chemical treatment on LOX, PIN II, and P4 transcript abundance in tomato foliage Treatment Jasmonic acid Arachidonic acid Benzothiadiazole P. syringae pv. tomato P. infestans H. zea feeding M. euphorbiae\ M. persicae feeding

LOX

PIN II

P4

j jjj 0 j jj j jj

jjj j\k 0 jjj j\k jjj 0

0 jjj jjj jjj jjj j jjj

jjj : strong induction. jj : modest induction j : weak induction. j\k : little or no induction. 0 : no effect.

produced in a plant by a biological inducer. Rather, differences in responses to different types of biological inducers result from differences in the combinations of signals released by the various inducing agents, irrespective of their taxonomic identity [35, 53 ]. An aphid, for example, feeds by extending its proboscis intercellularly to phloem tissue, and may at the same time release a battery of hydrolytic enzymes and\or plant growth regulators [1, 33, 38 ]. This combination of physical and chemical traumas is probably similar in subtlety to the initial damage during penetration of a fungal hypha into a leaf with the concomitant release of hydrolytic enzymes. Indeed, it is very different from the shearing and crushing of cells that occurs when a chewing insect feeds on a leaf. With respect to chemical signaling, the results presented here agree with the dichotomy of signaling discussed earlier (Fig. 1), where JA induces local responses associated with insect injury and wounding, and AA elicits local responses typically associated with pathogen infection. AA did not induce PINII in tomato, consistent with a previous report [22 ]. BTH induced PR-protein expression, but not PINII, LOX, or necrosis at concentrations that induce disease resistance. The pattern of gene expression observed in our study corroborates the reported effects of salicylate and its analogs in other plants at SAR-inducing concentrations [26 ], but also distinguish salicylate’s effects from those induced by AA and other elicitors. Application of BTH to tomato leaves induced resistance to Pst, but not to P. infestans, two pathogens that produce very different lesion-types and symptoms, and that deploy different pathogenicity mechanisms to colonize their hosts. This further illustrates differences in vulnerability among pathogens to salicylate or to specific components of the SAR response induced by salicylates or salicylate mimics. Combinations of BTH and JA treatments showed the interaction of the two pathways engaged by these two chemicals. Our results demonstrate that activation of one pathway may suppress the other pathway, since P4 transcript accumulation was diminished by BTH\JA in comparison to the levels induced by BTH alone, and PINII levels were diminished by BTH\JA, relative to the levels induced by JA alone. The mechanism for this interaction is unresolved, although salicylates and related

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compounds can interfere with the activity of heme-containing oxidoreductases, such as the allene oxide synthase involved in JA biosynthesis [19, 42 ]. The response of tomato to JA and BTH contrasts with that recently reported in graminaceous monocotyledons. In rice, JA and 2,6-dichloroisonicotinic acid, another synthetic salicylate mimic, appear to be synergistic signaling compounds [49 ]. The rice blast fungus Magnaporthe grisea is capable of inducing a number of JA-inducible genes, some of which encode PR-proteins [50 ]. Both compounds induce LOX activity in rice [46,49 ] and can afford protection against pathogens in barley [37,48 ] and rice [49 ]. Collectively, these studies urge caution in seeking generalizations about jasmonate effects in plants, and suggest greater overlap in octadecanoid and salicylate signaling in monocotyledons than in dicotyledons. The capacity for Pst to rapidly engage signaling pathways associated with both insect and pathogen responses raises intriguing questions. Do the bacteria secrete different elicitors to induce both response pathways ? Does the bacteria-induced PINII accumulation arise through a systemin-jasmonate dependent signaling pathway or through a different pathway ? Are the intracellular pools of salicylic acid that presumably accumulate to induce PR-proteins in bacteria-challenged plants insufficient or sequestered to prevent interaction with targets in the signaling pathway leading to proteinase inhibitor accumulation ? Pathogen and plant-response mutants will be important biological reagents to begin to address these questions, as will the biochemical and genetic characterization of sites of interaction in signaling pathways involving salicylate and jasmonate. LOX expression in tomato leaves following challenge with the different agents indicate that this response is rather nonspecific. An earlier study showed that AA or its metabolites are released into potato leaf tissue by fungal spores about 9 to 12 h after inoculation [44 ], a period when LOX transcript abundance and activity begin to increase [25 ]. LOX activation, then, is among the earliest of the leaf responses to infection by P. infestans. However, the induced LOX does not appear to be critical for SAR against Pst or to other pathogens, since BTH alone had no detectable effect on LOX transcript levels (Fig. 7). High levels of LOX transcripts were detected in leaves following methyl jasmonate treatment in intact tomato plants, using a tomato leaf LOX probe [32 ]. Although the probe used in our study only shares 52 % overall homology with that sequence, the pattern of expression detected with the two probes was similar. In conclusion, the results of our study indicate that the model depicting distinct signaling for insect and pathogen responses in tomato is simplistic and not generally supported by the experiments with the different agents investigated. With respect to insect-plant and pathogen-plant interactions, responses may be agent-specific or may overlap in certain cases. However, chemical induction of disease resistance through salicylate-based chemistry suppresses jasmonate-dependent PINII expression. In a companion study [56 ], we show that tomato plants expressing chemically-induced SAR have depressed proteinase inhibitor and polyphenol oxidase activities, with a corresponding enhancement of insect herbivory. We thank Charles S. Gasser, Charles A. Ryan, and Jan A. L. van Kan for plasmids containing sequences for use as hybridization probes, Bryce W. Falk for providing

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