Proc. Natl. Acad. Sci. USAVol. 91, pp. 9799-9802, October 1994Plant Biology

Overexpression of the prosystemin gene in transgenic tomatoplants generates a systemic signal that constitutively inducesproteinase inhibitor synthesisBARRY McGuRL, MARTHA OROZCOCARDENAS*, GREGORY PEARCE, AND CLARENCE A. RYANtInstitute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

Contributed by Clarence A. Ryan, June 13, 1994

ABSTRACT Tomato plants (Lycopersicon esculentum,var. Better Boy) were stably transformed with a geneco tof the open reading frame of a prosystemin cDNA under theregulation of the cauliflower mosaic virus 35S promoter. Theleaves of the transgenic plants constitutively produced protein-ase inhibitor I and II proteins, which accumulated over time tolevels exceeding 1 mg/g of dry leaf weight. This phenotypecontrasts with that of untransformed plants, which produceproteinase inhibitor proteins in leaves only in response towounding or chemical inducers. The transgenic plants werealso stunted, although they appeared normal in all otherrespects. Grafting the upper half (scion) of an untransormedtomato plant onto the lower half (root stock) of a tomato plantexpressing the prosystemin transgene resulted in the constitu-tive expression of proteinase inhibitor proteins in the leaves ofboth the transformed root stock and the untransformed scion,demonstrating that expression of the prosystemin trangnegenerates a mobile wound signal. These results show thatsystemic signal propagation in the bransgenic plants does notrequire wounding, and they support the proposed role ofsystemin as the mobile wound signal.

Proteinase inhibitor proteins are produced by plants in re-sponse to pest or pathogen attacks (1) and have been shownto have a defensive role against herbivorous insects (2, 3). Intomato plants (Lycopersicon esculentum), members of twoproteinase inhibitor families, inhibitor I and inhibitor II, aresynthesized both locally and systemically in leaves in re-sponse to mechanical wounding-for example, the damagecaused by chewing insects (1). The systemic response in-volves a complex signaling pathway that is initiated by therelease of a mobile signal from the wound site (4).

Several mobile wound signals have been proposed, includ-ing abscisic acid (5), oligouronides (6), electrical activity (7),and an 18-amino acid polypeptide called systemin (8), whichis derived from a 200-amino acid precursor called prosys-temin (9). The polypeptide is a primary candidate for the roleof systemic signal for several reasons: it activates the ex-pression of inhibitor I and II genes when supplied to excisedyoung tomato plants; when applied to the wound site, radio-labeled systemin moved out of the wounded leaf at approx-imately the same rate as the endogenous wound signal andcould be recovered from phloem exudates within this timeframe (8); and an antisense prosystemin gene expressed intransformed tomato plants severely reduced systemic inhib-itor I and inhibitor II synthesis in response to wounding (9).

In this report we show that expression of a prosystemintransgene in tomato plants generates a systemic signal, whichconstitutively induces the synthesis of high levels of protein-ase inhibitor proteins in unwounded leaves. Untransformedscions that were grafted onto transgenic root stocks consti-

tutively synthesized large amounts of proteinase inhibitors Iand II in response to the prosystemin transgene expressed inthe root stocks. These results support the proposed role ofsystemin as the mobile wound signal.

MATERIALS AND METHODSConstruction of a Prosystemin Transgene. The 5' end of an

800-bp fragment ofa prosystemin cDNA (9), encoding aminoacids 16-200 of prosystemin, was ligated to a double-stranded oligonucleotide encoding the first 15 amino acids ofprosystemin plus 15 bases of the 5' untranslated region. Theresulting fragment was inserted into the polylinker of pBlue-script KS (+) (Stratagene), and DNA derived from a singlerecombinant was digested with HindIII (5' end) and Ssp 1 (3'end), generating a cDNA fragment which encoded the com-plete prosystemin open reading frame plus 15 bp of the 5'untranslated region and 103 bp of the 3' untranslated region.This fragment was cloned in the sense orientation relative tothe 35S cauliflower mosaic virus (CaMV) promoter within thebinary vectorpGA 643, which had been digested with Hind1IIand Hpa I.Tomato Transformation. The prosystemin expression con-

struct was introduced into Agrobacterium tumefaciens strainLA 4404 and was used to transform tomato (var. Better Boy)cotyledon tissue as previously described (10).

Grafting Experiments. Plants (Better Boy) were used 5-6weeks after germination. The upper halves of the plants wereexcised at the midpoint of the stem and all the leaves weretrimmed away with the exception of the pair of leavesimmediately beneath the apical meristem. The cut ends ofthestems were notched and grafting was accomplished by align-ing the notched ends of the stems and wrapping the graft sitewith Parafilm. Grafted plants were enclosed in a transparentplastic bag in the laboratory for 4 or 5 days before beingallowed to regenerate and grow in the greenhouse for 7-8weeks. Senescent leaves on the lower half of each graftedplant were periodically removed, although the plants wereleft undisturbed for at least 1 week prior to assaying the levelsof proteinase inhibitors.

Plant Growth Conditions. Plants were grown in controlledenvironment chambers at 270C with 17 hr of light [2504Em'2-s-'; 1 E (einstein) = 1 mol of photons] and 7 hr ofdarkness.Wounding and Immunoiogical Assay of Proteinase Inhibi-

tors. Wounding experiments utilized 14- to 16-day-old plants,which were wounded on the lower of the two primary leavesand left under constant illumination for 24 hr. The levels ofproteinase inhibitor I and inhibitor II were measured asdescribed (11, 12).

Abbreviation: CaMV, cauliflower mosaic virus.*Present address: Unidad de Investigacion en Biotecnologia Agri-cola, Corporacion Colombiana de Investigacion Agropecuaria,Apartado Aereo 151123, El Dorado, Bogota, D.C., Colombia.tTo whom reprint requests should be addressed.

9799

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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DNA and RNA Analysis. Nick-translation was performedaccording to the instructions of the kit manufacturer (Du-Pont). Hybridizations were carried out at 42°C in 50% (vol/vol) formamide as described (13). Blots were washed in 1xSSC/0.1% SDS at 650C.

RESULTS AND DISCUSSIONTo determine if increased levels of the systemin precursorwould enhance the signaling capability of plants whenwounded by chewing insects, a prosystemin transgene con-sisting of a cDNA fragment encoding the complete prosys-temin open reading frame driven by the 35S CaMV promoterwas used to stably transform tomato plants. Fifteen indepen-dent, primary transformants (Tj generation) were regener-ated and self-fertilized to produce the T2 generation. Incontrast to the expected result, that the expression of thetransgene would not directly induce the inhibitor proteins butwould enhance the signaling capacity of the plants whenwounded, 14 of the 15 T2 populations constitutively ex-pressed proteinase inhibitors. Proteinase inhibitors are notnormally expressed in young tomato leaves except in re-sponse to wounding or chemical inducers of proteinaseinhibitor synthesis.One of the highest expressing T2 transgenic populations

was chosen for detailed study and was used for all of theexperiments reported herein. Southern blot analysis revealedthat this line of plants contained at least three copies of theprosystemin transgene (data not shown). A transgene-specific probe hybridized to anmRNA band ofapproximately1 kb on a Northern blot of total RNA extracted from atransgenic plant (Fig. 1A, lane 2). This mRNA species wasnot present in RNA extracted from an untransformed plant(Fig. 1A, lane 1).

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An estimate of the relative levels ofprosystemin mRNA inthe transgenic plants and in untransformed plants was ob-tained by using the prosystemin cDNA to probe a Northernblot of total RNA samples extracted from five transgenicplants and from five untransformed plants (Fig. 1B). Thetransgenic prosystemin mRNA and the endogenous prosys-temin mRNA could not be distinguished on the basis of size,and both appear as a single band of approximately 1 kb on aNorthern blot. While the levels of prosystemin mRNA werenot quantified, it is apparent that the levels in the transgenicplants (Fig. 1B, lower gel) are much higher than in theuntransformed, control group (Fig. 1B, upper gel).

Fig. 2 shows the constitutive and wound-induced levels ofproteinase inhibitors in the leaves of 14-day-old tomato plantsexpressing the prosystemin transgene. Prior to wounding, thetransgenic plants expressed inhibitor I at a mean level of 87± 7 pg/ml of leafjuice and inhibitor II at a mean level of 75* 7 ,ug/ml of leaf juice, while the control plants did notexpress either proteinase inhibitor (Fig. 2 Top). In responseto wounding, the levels of inhibitors I and II increasedapproximately 2.5-fold in both the wounded and oppositeleaves of the transgenic plants (Fig. 2 Middle and Bottom).Expression ofthe prosystemin transgene does not, therefore,maximally induce proteinase inhibitor synthesis in theserelatively young tomato plants, since wounding further in-creased the levels of proteinase inhibitors.Measurement ofproteinase inhibitor I and II proteins in the

transgenic plants over a period of 6 weeks after plantingrevealed that the levels of both inhibitors increased steadilyover time in the lower leaves (Fig. 3 Upper), while the levelsin the upper, younger, leaves changed little between 2 and 4weeks and then increased dramatically between 4 and 6weeks (Fig. 3 Lower). This sharp increase between 4 and 6

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FIG. 1. (A) Northern blot analysis of prosystemin transgeneexpression in tomato plant leaves. Lane 1, total RNA extracted fromthe leaves of an untransformed tomato plant. Lane 2, total RNAextracted from the leaves of a tomato plant transformed with theprosystemin transgene. In each case 5 ug of total RNA was elec-trophoresed on a 1.4% agarose gel in the presence of formaldehydeand blotted onto nitrocellulose. The blot was probed with a nick-translated 400-bp Kpn I-Sac I binary vector DNA fragment contain-ing the 3' terminator region of the transgene. (B) Northern blotanalysis of total prosystemin mRNA in the leaves of five untrans-formed tomato plants (wild type) and in the leaves of five tomatoplants transformed with the prosystemin transgene (transgenic). Ineach case 5 pg of total RNA from the leaves of each plant waselectrophoresed, blotted, and probed with nick-translated prosys-temin cDNA. All the samples were analyzed on the same blot.Numbers indicate individual plant extracts.

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FIG. 2. Constitutive and wound-induced levels of proteinaseinhibitors I and II in the leaves of plants transformed with theprosystemin transgene (trans.) and in the leaves of untransformedtomato plants (control). (Top) Constitutive levels of proteinaseinhibitors I and II. (Middle) Wound-induced proteinase inhibitor Iand II levels in the wounded leaf. (Bottom) Wound-induced protein-ase inhibitor I and II levels in the opposite leaf. Proteinase inhibitorvalues are the mean values for 16 plants +SEM.

Proc. Natl. Acad. Sci. USA 91 (1994)

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Proc. Natl. Acad. Sci. USA 91 (1994) 9801

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FIG. 3. Time course of accumulation of proteinase inhibitor I1(o)and proteinase inhibitor II (*) in the lower and upper leaves ofunwounded tomato plants expressing the prosystemin transgene.The upper leaves are those on branches arising from the upper 25%of the stem. The lower leaves are those on branches arising from thelower 25% ofthe stem. Each data point is the mean value for 12 plants±SEM. No proteinase inhibitor synthesis was detected in 12 un-transformed control plants.

weeks may be caused by the release of large amounts oftransgenic systemin from lower leaves, which begin to showvisible signs of senescence at this time.

In some transgenic plants the level of inhibitor II in theupper leaves at 6 weeks exceeded 1 mg/ml of leafjuice, threetimes higher than the highest level previously reported, whichhad been obtained by prolonged exposure oftomato plants tomethyl jasmonate, a chemical inducer of proteinase inhibitorsynthesis (14). The expenditure of energy and amino acidsrequired to continuously produce such large amounts ofproteinase inhibitors may account for the stunted appearanceof the transgenic plants (Fig. 4). It is also possible that thetransgenic plants are overexpressing other proteins which areregulated by systemin or by other biologically active peptidesderived from prosystemin. These plants may, therefore,

prove to be an invaluable resource for identifying, otherproteins which are regulated by a prosystemin-dependentsignaling pathway.The observation that tomato plants expressing the prosys-

temin transgene constitutively synthesize proteinase inhibi-tors was surprising, since the low level ofconstitutive prosys-temin expression normally observed in untransformed to-mato plants regulates the wound-induced expression ofproteinase inhibitors (9) but is not associated with constitu-tive synthesis of proteinase inhibitors. We propose thatprosystemin is normally synthesized and then processed, andthe mature systemin is sequestered until it is released inresponse to wounding. The relatively large amount of sys-temin produced in the transgenic plants may saturate thestorage mechanism, resulting in the continuous release ofsystemin from the cells in which it is synthesized.On the other hand, it is possible that prosystemin and the

processing enzyme(s) responsible for releasing systemin maybe produced in different cell types, mixing of the two nor-mally occurring as a consequence of wounding. Transgenicprosystemin production is under the control ofthe 35S CaMVpromoter, which should be active in every cell type, includingcells producing processing enzyme(s). In this case, woundingwould not be required to expose prosystemin to the process-ing enzyme(s) and systemin would be continuously released.Another, related, possibility is that the synthesis of prosys-temin may be cell-type-specific, and the expression of thetransgene in cells throughout the plant might result in abnor-mal processing and release of systemin, involving proteinasesto which prosystemin is not normally exposed. A betterunderstanding of the normal fate of prosystemin will berequired to fully explain the phenotype of these transgenicplants.We were unable to directly test our assumption that tissues

expressing the prosystemin transgene continuously releaseprocessed systemin, as we do not yet have a reliable assay tomeasure the level of systemin in plants. We were able toindirectly test this assumption, however, by performing asimple grafting experiment to demonstrate that tissue ex-pressing the prosystemin transgene releases a mobile woundsignal. The upper halves of three untransformed tomatoplants were removed and were used as scions for graffingonto the lower halves (the root stock) of three tomato plantsexpressing the prosystemin transgene. As controls, two

FIG. 4. Photograph showing the relative sizes of two tomato plants expressing the prosystemin transgene (right) and two untransformedtomato plants (left).

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Table 1. Proteinase inhibitor expression in grafted tomato plantsProteinase inhibitor, Mg/ml of leafjuice

In scion In root stock

Plant Scion Root stock Inh I Inh H Inh I Inh II1 Wild type Transgenic 159.3 ± 17.1 337.8 ± 44.7 77.8 ± 22.7 76.0 ± 14.52 Wild type Transgenic 109.5 ± 28.2 193.3 ± 48.7 271.7 ± 21.4 405.5 ± 23.43 Wild type Transgenic 220.8 ± 36.3 262.0 ± 43.2 273.7 ± 30.7 375.0 ± 48.14 Wild type Wild type 13.8 ± 6.7 7.8 ± 2.6 15.5 ± 6.7 6.5 ± 2.95 Wild type Wild type 5.5 ± 2.7 7.2 ± 2.7 3.2 ± 1.4 4.7 ± 2.1The upper halves (scions) of three untransformed tomato plants were grafted onto the lower halves (root stock) of three

tomato plants expressing the prosystemin transgene (plants 1, 2, and 3). As controls, untransformed scions were graftedonto untransformed root stock (plants 4 and 5). Each value is the mean proteinase inhibitor (Inh) concentration of six leaves+ SEM.

untransformed scions were grafted onto untransformed rootstock.

In every case the transgenic root stock systemically in-duced high levels of proteinase inhibitors in the untrans-formed scion (Table 1, plants 1, 2, and 3). In two of the threeplants (plants 2 and 3) higher levels of proteinase inhibitorswere found in the leaves of the transgenic root stock than inthe untransformed scions, while in plant 1 leaves of theuntransformed scion produced the higher levels ofproteinaseinhibitors. The control plants (4 and 5) showed a low level ofproteinase inhibitor synthesis in the leaves.The experiments reported in this paper demonstrate that

expression of a prosystemin transgene in tomato plantsresults in the generation of a systemic signal that inducesconstitutive proteinase inhibitor synthesis in the leaves.Oligouronides released from plant cell walls have been pro-posed as mobile signals (6), but they are now known to beimmobile over long distances (15). Abscisic acid has alsobeen proposed as a systemic signal (5), but its role in thesignaling pathway is still unresolved (16). The proposedinvolvement of electrical signals is largely based on a corre-lation between mechanical wounding and the induction ofelectrical activity (7), but no evidence has been presented tocausally relate wound-induced propagated electrical activityto the induction of proteinase inhibitor synthesis.The grafting experiment reported in this paper showed that

the expression of a prosystemin transgene generates a sys-temic signal that can be propagated over long distances in theabsence of wounding, thereby demonstrating that systeminplays a central role in the long-range induction of proteinaseinhibitors. This result, taken together with the known mo-bility of systemin within the phloem (8) and the observationthat an antisense prosystemin gene greatly reduces the sys-temic wound-induction of proteinase inhibitor synthesis (9),is most consistent with the proposed role of systemin as theprimary mobile wound signal.

We thank Mr. Greg Wichelns for growing the plants and Dr. M. L.Kahn for help with preparation of the figures. We also thank theBiomedical Communication Unit for their photographic work. Thisresearch was supported in part by Washington State UniversityCollege of Agriculture and Home Economics Project 1791 andNational Science Foundation Grants IBN-9104542 and IBN-9117795.

1. Ryan, C. A. (1990) Annu. Rev. Phytopathol. 28, 425-449.2. Hilder, V. A., Gatehouse, A. M. R., Sheerman, S. E., Barker,

R. F. & Boulter, D. (1987) Nature (London) 330, 160-163.3. Johnson, R., Narvaez, J., An, G. & Ryan, C. A. (1989) Proc.

Nati. Acad. Sci. USA 86, 9871-9875.4. Ryan, C. A. (1992) in 10 Years ofPlant MolecularBiology, eds.

Schilperoort, R. & Dure, L. (Kluwer, Boston), pp. 123-134.5. Pefia-Cortes, H., Sanchez-Serrano, J. J., Mertens, R., Wilm-

itzer, L. & Prat, S. (1989) Proc. NatI. Acad. Sci. USA 86,9851-9855.

6. Bishop, P. D., Makus, D. J., Pearce, G. & Ryan, C. A. (1981)Proc. Natl. Acad. Sci. USA 78, 3536-3540.

7. Wildon, D. C., Thain, J. F., Minchin, P. E. H., Gubb, I. R.,Reilly, A. J., Skipper, Y. D., Doherty, H. M., O'Donnell, P. J.& Bowles, D. J. (1992) Nature (London) 360, 62-65.

8. Pearce, G., Strydom, D., Johnson, S. & Ryan, C. A. (1991)Science 253, 895-898.

9. McGurl, B., Pearce, G., Orozco-Cardenas, M. & Ryan, C. A.(1992) Science 255, 1570-1573.

10. Narvaez-Vasquez, J. (1991) Ph.D. thesis (Washington StateUniv., Pullman).

11. Ryan, C. A. (1967) Anal. Biochem. 19, 434-440.12. Trautman, R., Cowan, K. M. & Wagner, G. G. (1971) Immu-

nochemistry 8, 901-916.13. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab.Press, Plainview, NY), 2nd Ed., pp. 9.0-9.62.

14. Farmer, E. E. & Ryan, C. A. (1990) Proc. NatI. Acad. Sci.USA 87, 7713-7716.

15. Baydoun, E. A.-H. & Fry, S. C. (1985) Planta 165, 269-276.16. Hildmann, T., Ebneth, M., Pefia-Cortes, H., Sanchez-Serrano,

J. J., Wilmitzer, L. & Prat, S. (1992) Plant Cell 4, 1157-1170.

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