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letters to nature IANWOIllly.T A. & Pond. I. L. Ardusbactaial ether lipids and chtmotaxonomy Syst Ang In response to plant defences, herbivores increase their pro- MiroMot 7, 253-25711986/ It Ungworthy, T A.. Ifolzer. G, Ze&us. I. G. a Tomabent. T. G. bo. and ameiso.branchoi glycerol duction of enzymes that detoxify allelochemicals, including dietha9 of the thermophix anaerobe Themankrulitthearnon etwourrec Syne.. App! Alarattol 4. cytochrome P450s (refs 15, 16). But herbivores are potentially 1-17119831 vulnerable to toxic allelochemicals in the duration between If Huber, It a al Formation of ammonium from nitrate during thernobthmuttoropha growth of the exocmdy thamophilic bacterium Ammaerelcs *soon gen now. p. now. Sat Appl Morolnal 11, ingesting toxins and induction of detoxification systems. Here 10-49 (156/61. we show that the corn earworm Helicoverpa sea uses jasmonate 20. Ilulut. It. a el. Aviles pyraphilm. new genus AnVspenes. repiesetLe • novel poop of wane and salicylate to activate four of its cytochrome P450 genes that hmenhamophilic halrogewouduing bateau Sere App! Aftonbset IS. 310-331 (19911 IL I)Rou. Al...y Id.trobatege of Farrow favnoserteenseniel m Pinreasefor tfrearketeLyy wile Da are associated with detoxification either before or concomitantly Costa. M. 5..1Xuat. J. C. & Williams. R. A.11/ 167-173 (Elsevier. London 1989). with the biosynthesis of allelochemicals. This ability to 'eaves- 22. Vanden Mumberg.I.0 AL. 'Mama. A I. Al. & KOMI>. W. N.7he cuente of bang eureinophdie the ink el the unique achand membrane hinds. EstionapAilcs 2. 161170 (19981. drop' on plant defence signals protects H. sea against toxins U. KOMI.. R. grpionta.11. & Runt:mm.1 Alonoalkyletha plimpholipals in 'hemline. produced by host plants. reducing lumen. Lkssallossmarta rundults and DesulforlistIslus an•vomc Anil, Altaand 176. The corn earworm, H. tea, is broadly polyphagous, with over IGO 435-412(2001). known host plants including herbs, shrubs and other low-lying 24. Mehta. G..v at Qum for bights laddereanesi Olisomenzation of a c)cbbutadsenedeniume. Mae.. Can,, Mr. El 11.1488-1490(19911. vegetation. We chose H. sea to address the general issue of whether n. SUMS. M.. Benne:1.11. Rumen. 1G. &)nien. AI. S. M.'lhe sequencing batch macaw as • powiful herbivorous insects can activate their enzymes that metabolize cud ins the study of slowly gamin anaerobic aransoniumaudinng nueromganissur. Appt Maemeirt Binierimol SO. M9-596119981 16 llunidrup, R & Dalsgaard.7 Production of Na through anaerobic ammonium oxidation couckd to nitrate reduction in marine sediments Ang Easton. Murano' 64 1312-1318 (20021 a CYP6828 in midgut CYP6828 in fatbody 7'7 What. /.. de Vries. S. Kuenen. I. G. & lawn. Al. S. AI. Imedairnem of a novel hydroulamine 10 10 ./odoreductuve in anaerobic ammonium oxidation. Rtorfrarirray39. 5105-541.1(2000). c 8 , 8 .an [Win. A. C T a Laner. S. A computanorol chemical study of penetration and displacement of 9 waiter films nen mineral surfaces Gmcleem Trans 0061P:01). .o g 6 6 Ala 29. Ratner-n.11.1 C. Pooma. I P 31. van Guntartn. w. F. DiNola. A. & Hoak. I. R. Molecular dsrumics with coupling man <111O7UI bath 1. Chow. Phys. 81. 34+4-3690 (1981). ▪S 4 .e 4 tai 2 §. 2 SOPPlemeaLaY 1010111141100 6. 0ImPimics the paper on N811014 wth2le 0 0 x Mtp:fiuwanalure.cOmMillut81. D 4 'A a Acknowledgemee% We thank I. G. Kucnen. II. I licnntra. S. Schouten and W Konings for CYP688 in mtdgu1 CYPE88 in fatbody 10 10 stimulating db.:M.1O114.0 Erkelens (University of Leiden) for access to the 600- and 750-MI Ix NAIR instruments.). A. Fuerst for cells of Gemmel.' obscurigiots“ and Pirellida sp. and training of 8 c 8 LA.y.N.. A1. wolicrs.Arts for help with electrum microscopy. and K. T. van de Pas.Schoonen for help with immunolluorescence. 6 6 5 4 • 4 Caimpeelna nada Material The authors declare that they have no competing financul 0 ts. 2 2 interest, 0 0 Corespondents and requests for materials should be addressed to z a in: .!..risteatmunl). 4 g EF-1u C11, 6628 ma large cyp6B8[ band Small b d Jasmonate and salicylate induce b CYP6a9 in midgut • 10 CYP6827 in midgut expression of herbivore 10 aril 5 7.5 cytochrome P450 genes O 7.5 .? 5 • 5 Xianchun Li' , Mary A. Schuler; 8, May R. Berenbauntt I la 1 210095. (Atm PI °tertian. Nanjing Agrieultoral LiniVaSity. Nanjing t Department of Entomology and Department of all and Structural Biology. LL 2.5 0 n ICI §. 2.5 0 Z 4 A University of Illinois. Urbana. Illinois 61801, USA Jasmonate and salicylate are plant-produced signals that activate CYP689 CYP6827 plant defence genes after herbivory" or pathogen' attack. Amplification of these signals, evoked by either enemy attack or experimental manipulation, leads to an increase in the syn- EF-lea EF•tu thesis of toxic compounds (allelochemicals)' and defence pro- teinr ° in the plants. Although the jasmonate and salicylate Floret CYP68 gene egression in H. zea in response to jasmonate (JA) and salicylate signal cascades activate different sets of plant defence genes10, or (SA). a. CYP688/CYP6828. b. CYP68.0. c. CYP6827. Total midgut or lathed/ MIAs from even act antagonistically''''', there is substantial communication the different diet treatments control diet. JA-L 2.9 nag "l a JA-fl. 290 pg g- r JA: between the pathways"". Jasmonate and salicylate also contrib- SA.L. 12 mpg - ' SA: SA.H. 1.2 mgg -1 SA) were separately amplified by RT-PCR and ute to protecting plants against herbivores by causing plants that analysed as described in Methods. The average fcid Indiction and standard deviations experience insect damage to increase their production of volatile (error bars) for three independent RT-PCR amplificationsare shown In the histogramt an molecules that attract natural enemies of herbivorous insects". autoradlogram of a representative blot Is shown below. 712 0 2002 Nature Publishing Group NATURE IAOL 419 117 OCTOBER 2001Iwyny.nature.comauture EFTA00765611  letters to nature Tate 1Effects of )aamonate and salltylate exposure on growth rate, welgM gain and mortality of H. rea Winton ot fourth War Marton of Win Maar Mcgtally on clay 3 Weight gain on thy 3 Pupal weight EMS fatelahly final au:orlon rate Tigatmente (c9 psi (m9) Mfg (74i (501 No earaure Contra tIbba 1.5 ±0.5 d 5.5 -z 0.5 OD 0.0b 384.4 t- 25.6 a 345.4 ± 13.5a 15.6 ± 5.1 0 84.3 ± 15.1 abc XanlhotoxIn diet 3.8 ± 0.8 b 11.7=0.3b 8.9 ± 1.9b 1022 t- 28.3 be 225.3 ± 18.5 b 30.0 ± 10.0 bed 70.0 ± 10.0 al Celery lenge 5.7 ± 0.3 a 14.7 -z 0.6 a 33.3 ± 15.3a 47.6 t- 2.7d 163.2 ± 23.4C 85.7 ± 11.8a 12.8± SS e JAL Control dials 1.5 ±0.5 d 5.5 -z 0.5e OD ± 0.0 b 3961 t- 7.8a 348.2 ± 24.3 a 5.7 ± 5.7 0 90.3 ± 5.8 a XantholoxIndlet 2.5!O.5O 9.5 -z 0.5c °At 0.0 b 78.9 t- 15.4 bad 2266 ± 2.5b Mit 11.8 bed 73.3_ 11.8 bog Celery leaves 2.5!O.5O 8.5 -z 0.5d 3.0 .±- 5.3 b 845 t- 17.9 bad 17A1 ± 39.5c 48.3 ± 16.1 be 48.7 ± 20.1 0 SAL Control Pets 1.5-10.5d 4.7 -z 0.6e &St- 5.8 b 382.1 ± 50.3 a 372.9 ± 11.5a 10.0 ± 10.0d 89.9 ± 10.0 ab Xantholoon del 2.5!O.5O 9.7 -z 0.6c OD t- 0.0 b 113.1 _9.3b 232.7 ± 5.5b 23.3 ± 5.8 ad 75.7 ± 5.8 tocl Celery leaves 2.5 ± 0.5 0 10.8 -: 0.3 b 7.0 ± 5.1 b 69.9 ± 15.5 ocl 1661 ± 15.6c 49.9 ± 10.0 b 50.0 ± 10.00 Efileds were meesurecl horn fourth Sir ta pay on cekry leaves and ce callid end 0.5% methateoin dela. The deb contained 2.9ICrJA OM) cr 12 eg ci“ SA (SA-40We es percentages %ere weave trarefamied tetare apart. %wile are the trdralslccrnecl means e.d. In eachmini. means (laved by cifferent Idlers are sisnficanly diluent < 0.05. nexilied L.SD blest). allelochemicals in response to the plant signal molecules jasmonate (AF102263), CYP6B9 (AF 140278), CYP6B27 (AF285829) and and salicylate before the accumulation of plant defence compounds. CYP6B28 (AF285186), in the midgut and fatbody—the principal Transcripts of four H. sea cytochrome P450 (P450) genes are sites of allelochemical detoxification in this species". inducible by furanocoumarins, chlorogenic acid, indole-3-carbinol Among the P450 transcripts examined, CYP6B8 and CYP6B28 and flavone16•", which suggests that these genes are involved in (99% amino acid identity), a pair of highly conserved paralogues", detoxifying a range of plant allelochemicals. But these four P450 were simultaneously amplified by polymerase chain reaction with genes are not universally inducible by all allelochemicals of host reverse transcription (RT—PCR), differentiated by digestion with plants; for example, gossypol, quercetin and rutin do not induce Xmul, and quantified by gel blot analysis (Fig. la). In midguts, their transcription". Baculovirus-mediated expression of one of CYP6B28 transcripts were induced about 5.0-fold by jasmonate and these proteins, CYP6B8, has shown that this protein can metabolize salicylate irrespective of the concentration, whereas CYP6B8 tran- xanthotoxin (221.1pmol per ml of baculovirus-expressing cell scripts were induced about 4.5-fold by either concentration of culture per min; unpublished data)—a furanocoumarin that is jasmonate, 3.3-fold by the low concentration of salicylate and 7.1- present in many host plants of H. sea and whose biosynthesis is fold by the high concentration of salicylate (Fig. la). In fatbody, stimulated by jasmonate and methyl jasmonate'. CYP6B28 transcripts were induced about 6.0-fold by either con- In Apium graveolem (celery)'•", a host plant of H. sea", xantho- centration of salicylate, 4.2-fold by the low concentration of toxin and the related furanocoumarin bergapten begin to accumu- jasmonate, and 6.2-fold by the high concentration of jasmonate; late after 24 h and reach maximal concentrations (representing a transcripts of CYP6B8 were increased to a lesser extent by jasmonate 40-70-fold increase) 4-6d after the application of jasmonate and and salicylate (Fig. la). methyl jasmonate''. Accordingly, we fed fifth instars of H. sea for The more divergent CYP6B9 and CYP6B27 transcripts derived 48 h with either artificial diets supplemented with jasmonate and from another pair of paralogous P450 genes (87% amino acid salicylate (at two concentrations for each chemical) or control diets, identity with CYP6B8), whose expression is restricted to midguts", and then examined expression of four P450 genes, CYP6B8 were separately detected by RT—PCR gel blot analysis (Fig. lb, c). In midguts, CYP6B9 transcripts were induced 6.0-fold by low concen- trations and 8.0-fold by high concentrations of jasmonate and salicylate (Fig. Ib), whereas CYP6B27 transcripts were induced 4.8-fold and 5.8-fold by low concentrations of jasmonate and - IT salicylate, respectively, and 6.9-fold and 7.8-fold by high concen- trations of jasmonate and salicylate, respectively (Fig. 1c). These results show clearly that expression of CYP6B is activated in the midgut and fatbody ofH. sea at the low concentrations of jasmonate and salicylate that are associated with pest damage and allelochemi- cal induction in its host plaints"•". To assess the specificity of this induction response, we tested further the induction of these P450 genes in response to two salicylate-related chemicals at equivalent concentrations to high concentration of salicylate. Methylparaben, which differs from salicylate in the position of its hydroxy group and in having an additional methyl ester group, did not induce any of the CYP6B genes examined. Not surprisingly, p-hydroxybenzoic acid, which differs from salicylate only in the position of its hydroxy group, acted as a weaker inducer than salicylate and increased the amounts nn 1 d NO of CYP6B8 and CYP6B28 transcripts roughly 2.0-fold, and the OOOO OOQC) QUO() UUUU amounts of CYP6B9 and CYP6B27 transcripts 4.0-5.0-fold (Fig. Control MP p-HBA SA-H 2). These results indicate that the degree of activation of H. sea Chemicals CYP6B genes by salicylate, jasmonate and related compounds is Ruin 2 C1P6Bexpressmn in response to salicylate (SA) and SA.retated dependent on structural features of these signal molecules. frhAroxybenzoic acid and methylparaben. Total FtNAs from catapillam fed on diets To test whether an increase in endogenous amounts of signal ambling 1.2 mg g "I SA (SA-Hr. 1.2 mg g-' methylparaben (MP) or 1.2 mg g I substances in plants that occurs before allelochemical biosynthesis is p-hAroxyl:enzcic acid (p-IIBA) were amplified by RT-PCR and analysed as described m sufficient to induce transcriptional expression, we allowed starved Methods. The relative induction and standard denabons fix three independent RT—PCR fourth instars ofH. zea to damage celery leaves and then determined amplifications are shown. the ability of these leaves to activate transcription of CYP6B in a XATURE j VOL 419 j II OCTOBER 2002 kveve.nature.cominature 0 2002 NaturePublishingGroup 713 EFTA00765612 letters to nature second set of fifth instars 2 and 4 h after damage. Compared with Reciprocal phenotypic responses characterize many antagonistic leaves from two undamaged control plants, which did not induce ecological interactions; if such reciprocal phenotypic change results CYP6B expression, leaves from all of the plants that had been from adaptive plasticity in the interacting species, then coevol- attacked for 2 and 4h induced expression of CYP6B28, CYP6B9 utionary interactions may result in the evolution not only of fixed and CY6B27 (Fig. 3). Analysis of these plants indicated that, in a adaptations but also of phenotypic plasticity". The induction of background of up to twofold constitutive differences in furanocou- P450 counterdefence genes in herbivores in response to plant signal marin content and composition among the test plants, no induced substances that are themselves inducible by herbivore damage might accumulation of furanocoumarin occurred. The differences in be an example of such phenotypic plasticity. Although it is well CYP6B expression were not correlated with variations in furano- known that herbivorous insects can enhance the expression of coumarin between plants (Fig. 3), which indicated that the acti- detoxification enzymes (counterdefences) in the presence of plant vation of CYP6B transcription resulted from an induction of allelochemicals (plant defences)1" 72121, we have shown here that H. jasmonate and/or other signal substances caused by the feeding zea responses to plant damage are more sophisticated than was behaviours of the first set of larvae. These data provide direct thought previously. By responding to plant signal molecules as well evidence that H. zea can intercept the plant defence signals elicited as the end-product allelochemicals, insects have the capacity to by its own feeding activity. equip themselves before (or concomitant with) the accumulation of To determine whether activation of P450 genes in advance of toxic concentrations of plant defence compounds. Although several exposure to furanocoumarins confers protection on H. sea, we examples have been found of plants using insect-derived signal compared the survival and growth of fourth instars that had prior substances to regulate their defence pathways'-', this represents to exposure to low concentrations of jasmonate and salicylate for 12 h our knowledge the first example of the use by insects of plant signal on celery leaves, 0.5% xanthotoxin diets, or control diets against molecules to regulate their defence systems against plant that ofcontrol larvae that had not been exposed in advance to signal allelochemicals. substances. Two-way analysis of variance (ANOVA) and multiple The ability to use plant signal molecules as cues for activating a comparison tests on mortality, weight gain, growth rate and pupa- detoxification system may be of particular value to a broadly tion success (Table 1) indicated that caterpillars exposed to jasmo- polyphagous herbivore such as H. sea. In contrast to oligophagous nate and salicylate survived better on celery leaves and 0.5% species, which encounter a relatively narrow and generally predict- xanthotoxin diets for all parameters. On control diets, however, able range of plant allelochemicals, generalized herbivores may there were no significant differences in all parameters among the encounter any of several biosynthetically distinct compounds three treatments. These results suggest that the 'signal-eavesdrop- depending on host plant choice". Few commonalities exist ping' capability provides H. sea with prophylactic protection among the biosynthetic pathways that generate these plant defence against plant defences at no additional cost to fitness in the absence compounds other than the fact that they share jasmonate or of plant defences. salicylate as initiating signals. The ability of a generalist to respond a so ❑ InVeratorin • SendaF400 sec ▪ Isopimpinellin ▪ xanthotoxin s Sphondin § 3 40 Ea Total u g '5 E U. c 20 0 0 I Plant A 0 Undamaged 2 h of damage 4h of damage b 15 ❑ CYP688 ❑ CYP6828 ECYP689 IF 10 • CYP6827 ctC 2 c2 0 5 0 Plant A D E F G H Undamaged 2 to of damage Shot damage Rgure 3 CYP6Bachvation by feeding de damaged celery leaves. a, Content of total and leaves that were previouslyundamaged or damaged for 2 or 4 h by starved loath linters individual furanocoumarins for each plant. b. Relative nduchan and standard deviations were amplified by RT-PCR and analysed as deserted in Methods. for three independent RT-PCR amplifications. Total RNAs from fifth inters fed fa 48h on 710 0 2002 NaturePublishing Group NATURE [VOL 419111 OCTOBER 200:1wvv.natuze.cominaturc EFTA00765613  letters to nature to these signals by upregulating several detoxification genes may pathways in Anilddrmis are esiamial for resistance to distinct microbial pathogens. Poe. Mul Ana Ser. USA 95. 15107-15111(19981. maximize its ability to counter its host's response to damage, IL Penton. C A.. LereandiensH. C. Enyedi. A I. & aalsdn 1. T Tobacco mimic nun inoculation irrespective of taxon. 0 inhibits smond.induad iasmonic acid.medimed rano/neswithin but not between plants Plana 209. 87-95 11999). Methods 12. fetlockC. W. a d. Inverse relationship between systemic Rushlike of plants to microorganisim and to meet !tallowy. Gent But 9017-320 (19991. Test insects IS. Stout.51.1.. Eidantsef. A. L.Duffey.S. S ft Rostock P. M.Sursil intaactions in pathogen and insect An insecticide-susceptible laboratory grain of H. sea. provided by B. R. Banido (Abbott muck. SySICMIC plam.inediated attractions between pathogens and herbivores of the tomato. Laboratories). was used in all studies. We kept insects in an insectary maintained at 28°C Lfrommuten (intim:am. Hewer. 3164 Haw Rola 54. 115-110 (19991. in a 16:8 h light:dark cycle on a sernisynthetic control diet containing wheatgenn". 14. Thalet.I. 8. )aunonmeanduableplarg Mann. came manned para.:nunsof batman. Nome 399. 686-618 (1999/. Signal chemical Induction treatment IS. Schuler. M. A. The role idemoduonse P450 nxinoogrumases in plant-insect interactions. Ham Artificial diets containing 2.9 or 290 lig g jasmonate ISigmal. 12 ass or 1.2 mgg Proud. 113, 1411-14191199). salicylate 199%. Aldrich). 1.2 mg g methylparaben I Sigma). or 1.2 mg g p. 16. Li. X.. Berenboum. M. R. &Schuler. NI. A Mokrube doning and e.qsreseion of CTP6BS: 4 hydroxybenzoic acid (Sigma) were pecmided to 30 newly moulted fifth instars. The low xandicanxikinduable rd.:chrome P450 cDNA from Hottorrenn ant WC. 81100,1 Md Btel .10. concenua 'ions of iasmonate and salicylate were selected on the basis of endogenous 75-8418700). amounts of immonate and salicylate found in the host plants of H. ear"' and the high I7. 1.i. X, Berenbouna.M. R. de Schulte. M. A. Plant alelocbanicalidiffermtially regulate Behooving rea concentrations were selected to maximize the likelihood ofdetecting an upper limit on the crtochrome P450 MO. WAY Met Bid II, 343-35112002). response. After 48 h. midguts and (Audits were dissected out and total RNAs were 18. Milusch.M.* Bolao3.W.Airborne methyl rasnxinatenimulates die biosyntheis of furanocounwrins isolated from each type of tissue using guanidine-11(A extractionv and then resuspended in the leaves of celery plants (Apinia awnvolnol. Egeerientie 52, 739-743 ( I 996) in diethyl pyrocarbonate I DE PC: • trea ted water. 19. ear. It M. AR Mika fork LarststhdLfilfinents. Lady Sat*. andHanelMatiorrmitorne alter Commitment Omni Sraresentl Canal. Wt l and 2 (A. C Allyn Mut Entomology. Sarasota. Horeb. 19721. Relay damage and Induction treatment 20. Li. X. Berenbaum.51. R & Schulte. Al. A. Cytochroene P45nand actin sum opened in Ilebeerogn We grew nine celery plants individually in pots under laboratory conditions for 3 weeks to au and Ikea spa Amgen paresis...366.43p tdentitionton. gar osneentent. and evolution. ensure that they were free ofherbivore and pathogen infestation. Eight of than were free of /men &atm MA But 12, 311-320 Ono*. infestation and were assigned randomly to one of duce groups: undamaged control. 2 h of It, Row. 1. re el Al111.411144410.1.11441 depletion of • potato lipoxygause seduces wound induction of damage and 4 h of damage. For each plant. four menu with hilly expanded pain of leaflets Nous:use inhibmin and increases weight gun of insect pots. Pew. Mid And. Sn. USA 96. and a terminal leaflet were choien for treatment.(hi each stem. WO fourth Instrs that had 1146-1151 05991. been started for 4 h were confined to the second pair of leafless by two small clip ages. 22. Amami. A. A Phenotypk plankity in the interactionsand evolution of species. Serowe2K 321-326 with one larva per leaflet. For the undamaged controls. clip cages without larvae were (20011. placed on the second pair of leaflets on each stein for 4 h. After damage treatments. the 23. Danielson. P. B.. Macintyre.R I. *Fogleman.) C. Molecular cloning of a family of senobitak- second pair of leaflets was removed from each treated stem. We used one damaged leaflet indualle drorophilid cytoduome P454* Evidence for imolwinent in host-plant alklothemkal to feed a newly moulted fifth instar that had been starved for 4 h. NE pooled another leaflet rokunce. Peer Abel Aid Set USA 94. 10797-10802 (1997). with the other three kJ nets from the same plant. oven-dried them at 50°C (or 24 h and 24. Snyder.ht L. Andersen. F ta Enenian.R. Eyre:don of cnochionw NiOgenes ofthe used them for furanocoumarin determination. After 48 h of feeding on the damaged CYP4 famdy in midgut and fatbody °Diu tobaccohonnowm. Alandimi texts An*. /Winn Blithp. leaves. lame were killed and the mdguts and fatbmIlt, were rerniwed.The midgets from 321. I3-Xi 11995). the four larvae kd the damaged leaVeN from the same plant were pooled together and total 25. Beembaum. M. R. an lamb, grelegy ofrke Them Roane Ids Puga.A.& Wallace. K. R1553-571 RNA was isolated as described'. Oh*, & Francs. Philadelphia. PA, 1999). 26. Waldbauer.G. P. Cohen. R. W. & Friedman.* An improved procedure fa laboratory rearing of the RNA and turanocounatin analysis corn noworm. Heliothis ma iLepadown• Nocundm). (emuLein Enramed 17.113-114 Mb*. We carried out RNA isolation and RT-PCR gel blot analyses as described". For each RNA 27. Sambrook. ). Enoch. E. F. & Mantis. T. 31nkeubv <Seeing. A 14Invesary Manuel 7.23-716 (Cold sample. three independent RT-N:R amplifications were carried out. For furanocoumarin Spring Harbor Laboratory Pane. Cold Spring limbo,. NY. 19894 assay. all leaf samples were weighed separately and ground to a fine powder with a plastic 28. Zanged. A R. Amu. A. XL & Rerenbaum. A R. Physsolopcal rue ofan inducedchemicaldefense rod inside Eppendod tubes. Furanocoumarins were extracted, separated and photosynthesis. respiration. biosynthesis, and pond. neceama 109. 413.441 119971. quantified as described". Acknowledgments 1* thank M.Catroll for valuable discussion. This researchwassupported by asmonate and salicylate protection bioassay gr.tro, from USIM to M.R.B. and NIA.S.. and from the China Natural Science Foundation to X.L. Newly moulted fourth mums 2701 from the University of Illinois laboratory colony were divided randomly into three groups (90 larvae per group) and reared individually in Compering Interests statement The authors declare that they have no competing financial plastic cups with fresh control diets or supplemented diets containing 7-9 pgg imern:s. jasmonate or I 2 pgg salicylate. After 12 h of exposure to plant signal molecules, each group was divided further into three subgroups (30 larvae per subgroup. three replicates of Correspondence .Ind requests for materials should be addressed to ARR. ID insects) that were transferred to plastic cups with fresh control diets. diets containing le. mad: maybo,ustse.edu) or XL. (e-mail IxoBlife.muc.eduk 0.5% xanthotoxin. or celery leaves. Initial weights were recorded for every individual. All larvae were weighed again on the third day after transfer to experimental diets to determine weight gain. We monitored survival and developmental stage daily until all larvae had either pupated or died. Differences in weight gain. duration of fourth and fifth instars. mortality. pupal weight and percentage of pupation among the treatments were evaluated by ANt/VA. followed by modified least significant difference test (LSD *test/. with the significance level set at P < 0.05 using the SAS statistics program. Received 19 March accepted 27 lune 2002: doi:10.1035/nature01003. A biological role for prokaryotic 1. McCloud. F. S. & Radio* 1. T Herbivoreand catapdlar reguinums amplify the woundanduring increases in pinwale acd but not nicotine in Nintione tylnsins. Hama 103.430-435'19971. CIC chloride channels L 11.1. L.. bluiphy. 5 It it klion.G. W. Dom salighc and an asasignalincotton foe induced resistance to 1164nnespr am? 1. Owe. gent 23.1005-1818 (1997). Ramkumar Iyer, Tina M. Iverson, Alessi° Accardi & Christopher Iii ller 3. Moran. P. & Thompson.G. A. Molecular responses to aphid feeding inMeltdown in relation to plant defense pode.vays. P444I Ansa 125. 1074-1085120011. Deporunent of Biochemistry, Howard Hughes Medical Institute, Brandeis 4. Setae. M.Schulaw. V. & Raskin. I. Endogenous =M)/ salierlace in Nthoinnkoculated tobacco University, 1Valthant, Massachusetts 02454, USA plants. Paine Paysial 11& M7-39211998). 5. gamine& M.Oldham. N. t & Baldwin. 1. T Rapid HPLC sateningof tomonaknwhiced increases sec tobacoaalkamds. phenolics. and deterpree glycosides in Ne4flatilt anounira.1. Apt. lined Chow. An unexpected finding emerging from large-scale genome ana- 49. 3553-3558 (20011. lyses is that prokaryotes express ion channels belonging to ▪ Tschamdm T. Thiessen. 5.. Dolch.R /4 Boland. W. Hatinon. induced resistance and manly,' molecular families long studied in neurons. Bacteria and archaea *MI transfer in Arniughernow Riodinii. Sawyer. Fed. 29. 1025-1047 (NOD 7. Volker. 5. kern. P * Boland. W Biosyrithek or furonacoumoriew menbanateindependent are now known to carry genes for potassium channels of the pienttation of umbellikione in Arnow prints, (Apiacciet Phinarlinnorry 50.1141-1145 09991 voltage-gated, inward rectifier and calcium-activated classes", & Kiddie. G. A. DoodWY. K I. & Walbgrear. R M saliejlic acid-induced accumulation or Clc-type chloride channels", an ionotropic glutamate receptor' glucosinotnes in oared rap (lbw vs wpm/Ukase./ Exp Botany 45. 1343-1)46115941. and a sodium channels. For two potassium channels and a 9. llialer.1. S. Mout.ht I.. Kansan. R &Duffey.5.5. Exogenous immonates nimobie insect wounding in 1011W10 plants apnyrntool onekamm) in tit< laknalogy and Inld./. Chem Ea U1767-178111990 chloride channel, these homologues have provided a means to to llsomma. B. P I. red &Wilt jaimanatedererdent arid sabeYbandrIsendan ckfewertsforne direct structure determination'". And yet the purposes of these NATUREI VOL 419117 OCTOBER 2002 1wvevanaturecominature 0 2002 NaturePublishing Group 715 EFTA00765614