Overexpression of the prosystemin gene in transgenic tomato plants

Proc. Natl. Acad. Sci. USA

Vol. 91, pp. 9799-9802, October 1994 Plant Biology

Overexpression of the prosystemin gene in transgenic tomato plants generates a systemic signal that constitutively induces proteinase inhibitor synthesis BARRY McGuRL, MARTHA OROZCOCARDENAS*, GREGORY PEARCE, AND CLARENCE A. RYANt Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

Contributed by Clarence A. Ryan, June 13, 1994

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

Tomato plants (Lycopersicon esculentum, ABSTRACT var. Better Boy) were stably transformed with a gene co t of the open reading frame of a prosystemin cDNA under the regulation of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants constitutively produced proteinase inhibitor I and II proteins, which accumulated over time to levels exceeding 1 mg/g of dry leaf weight. This phenotype contrasts with that of untransformed plants, which produce proteinase inhibitor proteins in leaves only in response to wounding or chemical inducers. The transgenic plants were also stunted, although they appeared normal in all other respects. Grafting the upper half (scion) of an untransormed tomato plant onto the lower half (root stock) of a tomato plant expressing the prosystemin transgene resulted in the constitutive expression of proteinase inhibitor proteins in the leaves of both the transformed root stock and the untransformed scion, demonstrating that expression of the prosystemin trangne generates a mobile wound signal. These results show that systemic signal propagation in the bransgenic plants does not require wounding, and they support the proposed role of systemin as the mobile wound signal.

MATERIALS AND METHODS Construction of a Prosystemin Transgene. The 5' end of an 800-bp fragment of a prosystemin cDNA (9), encoding amino acids 16-200 of prosystemin, was ligated to a doublestranded oligonucleotide encoding the first 15 amino acids of prosystemin plus 15 bases of the 5' untranslated region. The resulting fragment was inserted into the polylinker of pBluescript KS (+) (Stratagene), and DNA derived from a single recombinant was digested with HindIII (5' end) and Ssp 1 (3' end), generating a cDNA fragment which encoded the complete 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 to the 35S cauliflower mosaic virus (CaMV) promoter within the binary vector pGA 643, which had been digested with Hind1II and Hpa I. Tomato Transformation. The prosystemin expression construct was introduced into Agrobacterium tumefaciens strain LA 4404 and was used to transform tomato (var. Better Boy) cotyledon tissue as previously described (10). Grafting Experiments. Plants (Better Boy) were used 5-6 weeks after germination. The upper halves of the plants were excised at the midpoint of the stem and all the leaves were trimmed away with the exception of the pair of leaves immediately beneath the apical meristem. The cut ends of the stems were notched and grafting was accomplished by aligning the notched ends of the stems and wrapping the graft site with Parafilm. Grafted plants were enclosed in a transparent plastic bag in the laboratory for 4 or 5 days before being allowed to regenerate and grow in the greenhouse for 7-8 weeks. Senescent leaves on the lower half of each grafted plant were periodically removed, although the plants were left undisturbed for at least 1 week prior to assaying the levels of proteinase inhibitors. Plant Growth Conditions. Plants were grown in controlled environment chambers at 270C with 17 hr of light [250 4Em'2-s-'; 1 E (einstein) = 1 mol of photons] and 7 hr of darkness. Wounding and Immunoiogical Assay of Proteinase Inhibitors. Wounding experiments utilized 14- to 16-day-old plants, which were wounded on the lower of the two primary leaves and left under constant illumination for 24 hr. The levels of proteinase inhibitor I and inhibitor II were measured as described (11, 12).

Proteinase inhibitor proteins are produced by plants in response to pest or pathogen attacks (1) and have been shown to have a defensive role against herbivorous insects (2, 3). In tomato plants (Lycopersicon esculentum), members of two proteinase inhibitor families, inhibitor I and inhibitor II, are synthesized both locally and systemically in leaves in response to mechanical wounding-for example, the damage caused by chewing insects (1). The systemic response involves a complex signaling pathway that is initiated by the release of a mobile signal from the wound site (4). Several mobile wound signals have been proposed, including abscisic acid (5), oligouronides (6), electrical activity (7), and an 18-amino acid polypeptide called systemin (8), which is derived from a 200-amino acid precursor called prosystemin (9). The polypeptide is a primary candidate for the role of systemic signal for several reasons: it activates the expression of inhibitor I and II genes when supplied to excised young tomato plants; when applied to the wound site, radiolabeled systemin moved out of the wounded leaf at approximately the same rate as the endogenous wound signal and could be recovered from phloem exudates within this time frame (8); and an antisense prosystemin gene expressed in transformed tomato plants severely reduced systemic inhibitor I and inhibitor II synthesis in response to wounding (9). In this report we show that expression of a prosystemin transgene in tomato plants generates a systemic signal, which constitutively induces the synthesis of high levels of proteinase inhibitor proteins in unwounded leaves. Untransformed scions that were grafted onto transgenic root stocks consti-

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

The publication costs of this article were defrayed in part by page charge payment. 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 performed according to the instructions of the kit manufacturer (DuPont). Hybridizations were carried out at 42°C in 50% (vol/ vol) formamide as described (13). Blots were washed in 1 x SSC/0.1% SDS at 650C.

RESULTS AND DISCUSSION To determine if increased levels of the systemin precursor would enhance the signaling capability of plants when wounded by chewing insects, a prosystemin transgene consisting of a cDNA fragment encoding the complete prosystemin open reading frame driven by the 35S CaMV promoter was used to stably transform tomato plants. Fifteen independent, primary transformants (Tj generation) were regenerated and self-fertilized to produce the T2 generation. In contrast to the expected result, that the expression of the transgene would not directly induce the inhibitor proteins but would enhance the signaling capacity of the plants when wounded, 14 of the 15 T2 populations constitutively expressed proteinase inhibitors. Proteinase inhibitors are not normally expressed in young tomato leaves except in response to wounding or chemical inducers of proteinase inhibitor synthesis. One of the highest expressing T2 transgenic populations was chosen for detailed study and was used for all of the experiments reported herein. Southern blot analysis revealed that this line of plants contained at least three copies of the prosystemin transgene (data not shown). A transgenespecific probe hybridized to an mRNA band of approximately 1 kb on a Northern blot of total RNA extracted from a transgenic plant (Fig. 1A, lane 2). This mRNA species was not present in RNA extracted from an untransformed plant (Fig. 1A, lane 1).

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An estimate of the relative levels of prosystemin mRNA in the transgenic plants and in untransformed plants was obtained by using the prosystemin cDNA to probe a Northern blot of total RNA samples extracted from five transgenic plants and from five untransformed plants (Fig. 1B). The transgenic prosystemin mRNA and the endogenous prosystemin mRNA could not be distinguished on the basis of size, and both appear as a single band of approximately 1 kb on a Northern blot. While the levels of prosystemin mRNA were not quantified, it is apparent that the levels in the transgenic plants (Fig. 1B, lower gel) are much higher than in the untransformed, control group (Fig. 1B, upper gel). Fig. 2 shows the constitutive and wound-induced levels of proteinase inhibitors in the leaves of 14-day-old tomato plants expressing the prosystemin transgene. Prior to wounding, the transgenic plants expressed inhibitor I at a mean level of 87 ± 7 pg/ml of leaf juice and inhibitor II at a mean level of 75 * 7 ,ug/ml of leaf juice, while the control plants did not express either proteinase inhibitor (Fig. 2 Top). In response to wounding, the levels of inhibitors I and II increased approximately 2.5-fold in both the wounded and opposite leaves of the transgenic plants (Fig. 2 Middle and Bottom). Expression of the prosystemin transgene does not, therefore, maximally induce proteinase inhibitor synthesis in these relatively young tomato plants, since wounding further increased the levels of proteinase inhibitors. Measurement of proteinase inhibitor I and II proteins in the transgenic plants over a period of 6 weeks after planting revealed that the levels of both inhibitors increased steadily over time in the lower leaves (Fig. 3 Upper), while the levels in the upper, younger, leaves changed little between 2 and 4 weeks and then increased dramatically between 4 and 6 weeks (Fig. 3 Lower). This sharp increase between 4 and 6 25

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FIG. 1. (A) Northern blot analysis of prosystemin transgene expression in tomato plant leaves. Lane 1, total RNA extracted from the leaves of an untransformed tomato plant. Lane 2, total RNA extracted from the leaves of a tomato plant transformed with the prosystemin transgene. In each case 5 ug of total RNA was electrophoresed on a 1.4% agarose gel in the presence of formaldehyde and blotted onto nitrocellulose. The blot was probed with a nicktranslated 400-bp Kpn I-Sac I binary vector DNA fragment containing the 3' terminator region of the transgene. (B) Northern blot analysis of total prosystemin mRNA in the leaves of five untransformed tomato plants (wild type) and in the leaves of five tomato plants transformed with the prosystemin transgene (transgenic). In each case 5 pg of total RNA from the leaves of each plant was electrophoresed, blotted, and probed with nick-translated prosystemin 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 proteinase inhibitors I and II in the leaves of plants transformed with the prosystemin transgene (trans.) and in the leaves of untransformed tomato plants (control). (Top) Constitutive levels of proteinase inhibitors I and II. (Middle) Wound-induced proteinase inhibitor I and II levels in the wounded leaf. (Bottom) Wound-induced proteinase inhibitor I and II levels in the opposite leaf. Proteinase inhibitor values are the mean values for 16 plants +SEM.

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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 of unwounded 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 the lower 25% of the stem. Each data point is the mean value for 12 plants ±SEM. No proteinase inhibitor synthesis was detected in 12 untransformed control plants.

weeks may be caused by the release of large amounts of transgenic systemin from lower leaves, which begin to show visible signs of senescence at this time. In some transgenic plants the level of inhibitor II in the upper leaves at 6 weeks exceeded 1 mg/ml of leafjuice, three times higher than the highest level previously reported, which had been obtained by prolonged exposure of tomato plants to methyl jasmonate, a chemical inducer of proteinase inhibitor synthesis (14). The expenditure of energy and amino acids required to continuously produce such large amounts of proteinase inhibitors may account for the stunted appearance of the transgenic plants (Fig. 4). It is also possible that the transgenic plants are overexpressing other proteins which are regulated by systemin or by other biologically active peptides derived from prosystemin. These plants may, therefore,

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prove to be an invaluable resource for identifying, other proteins which are regulated by a prosystemin-dependent signaling pathway. The observation that tomato plants expressing the prosystemin transgene constitutively synthesize proteinase inhibitors was surprising, since the low level of constitutive prosystemin expression normally observed in untransformed tomato plants regulates the wound-induced expression of proteinase inhibitors (9) but is not associated with constitutive synthesis of proteinase inhibitors. We propose that prosystemin is normally synthesized and then processed, and the mature systemin is sequestered until it is released in response to wounding. The relatively large amount of systemin produced in the transgenic plants may saturate the storage mechanism, resulting in the continuous release of systemin 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 may be produced in different cell types, mixing of the two normally occurring as a consequence of wounding. Transgenic prosystemin production is under the control of the 35S CaMV promoter, which should be active in every cell type, including cells producing processing enzyme(s). In this case, wounding would not be required to expose prosystemin to the processing enzyme(s) and systemin would be continuously released. Another, related, possibility is that the synthesis of prosystemin may be cell-type-specific, and the expression of the transgene in cells throughout the plant might result in abnormal processing and release of systemin, involving proteinases to which prosystemin is not normally exposed. A better understanding of the normal fate of prosystemin will be required to fully explain the phenotype of these transgenic plants. We were unable to directly test our assumption that tissues expressing the prosystemin transgene continuously release processed systemin, as we do not yet have a reliable assay to measure the level of systemin in plants. We were able to indirectly test this assumption, however, by performing a simple grafting experiment to demonstrate that tissue expressing the prosystemin transgene releases a mobile wound signal. The upper halves of three untransformed tomato plants were removed and were used as scions for graffing onto the lower halves (the root stock) of three tomato plants expressing the prosystemin transgene. As controls, two

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

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

Table 1. Proteinase inhibitor expression in grafted tomato plants Proteinase inhibitor, Mg/ml of leafjuice In scion In root stock Inh I Plant Scion Root stock Inh H Inh I Inh II 1 Wild type Transgenic 159.3 ± 17.1 337.8 ± 44.7 77.8 ± 22.7 76.0 ± 14.5 2 109.5 ± 28.2 271.7 ± 21.4 Wild type Transgenic 193.3 ± 48.7 405.5 ± 23.4 Wild type 220.8 ± 36.3 262.0 ± 43.2 273.7 ± 30.7 3 Transgenic 375.0 ± 48.1 4 Wild type 13.8 ± 6.7 7.8 ± 2.6 15.5 ± 6.7 Wild type 6.5 ± 2.9 Wild type 5.5 ± 2.7 3.2 ± 1.4 Wild type 7.2 ± 2.7 4.7 ± 2.1 5 The 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 grafted onto 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 root stock. In every case the transgenic root stock systemically induced high levels of proteinase inhibitors in the untransformed scion (Table 1, plants 1, 2, and 3). In two of the three plants (plants 2 and 3) higher levels of proteinase inhibitors were found in the leaves of the transgenic root stock than in the untransformed scions, while in plant 1 leaves of the untransformed scion produced the higher levels of proteinase inhibitors. The control plants (4 and 5) showed a low level of proteinase inhibitor synthesis in the leaves. The experiments reported in this paper demonstrate that expression of a prosystemin transgene in tomato plants results in the generation of a systemic signal that induces constitutive proteinase inhibitor synthesis in the leaves. Oligouronides released from plant cell walls have been proposed as mobile signals (6), but they are now known to be immobile over long distances (15). Abscisic acid has also been proposed as a systemic signal (5), but its role in the signaling pathway is still unresolved (16). The proposed involvement of electrical signals is largely based on a correlation between mechanical wounding and the induction of electrical activity (7), but no evidence has been presented to causally relate wound-induced propagated electrical activity to the induction of proteinase inhibitor synthesis. The grafting experiment reported in this paper showed that the expression of a prosystemin transgene generates a systemic signal that can be propagated over long distances in the absence of wounding, thereby demonstrating that systemin plays a central role in the long-range induction of proteinase inhibitors. This result, taken together with the known mobility of systemin within the phloem (8) and the observation that an antisense prosystemin gene greatly reduces the systemic wound-induction of proteinase inhibitor synthesis (9), is most consistent with the proposed role of systemin as the primary 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 the Biomedical Communication Unit for their photographic work. This research was supported in part by Washington State University College of Agriculture and Home Economics Project 1791 and National Science Foundation Grants IBN-9104542 and IBN-9117795.

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