Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8273-8276, September 1993 Plant Biology Expression of an antisense prosystemin gene in plants reduces resistance toward Manduca sexta larvae (protelnae inhibitors/plant defense/trausgenic tomato plants/) MARTHA OROZCO_CARDENAS*, BARRY MCGURL, AND CLARENCE A. RYANt Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 Contributed by Clarence A. Ryan, June 15, 1993

ABSTRACT The growth rates of Manduca sexta (tobacco A model for the signaling of the inhibitor genes has been hornworm) larvae feeding on tomato plants constitutively presented (7, 8) in which systemin is released, as the result of expressing a prosystemin antisense gene were =3 times higher wounding by attacking or other mechanical damage to than growth rates oflarvae feeding on nontransformed control leaves, and is translocated throughout the plant where it plants. The levels of proteinase inhibitor I and inhibitor H interacts with receptors in the plasma membrane of both proteins in leaves of tomato plants expressing the antisense nearby and distal cells. It was proposed that the interaction prosystemin gene remained atundetectable levels until the sixth with receptors activates a lipase, releasing membrane- day of larval feeding and then increased throughout the plants derived linolenic acid into the , which is then to 100-125 pg/g of leaf tissue after 14 days. In control plants, converted to the powerful signaling molecule . levels ofproteinase inhibitor I and H proteins increased rapidly Jasmonic acid, or a derivative, is proposed to interact with from the second day of larval feeding and by the eighth day factors to activate the inhibitor genes (7, 8). containd levels of225 pg/g ofleaftissue and 275 pg/g ofleaf In this study, we investigate whether resistance can tissue, respectively, and then increased slowly thereafter. be affected by genetically modifying the function of a com- Prosystemin mRNA levels in antisense and control plants after ponent ofthe signaling system for the induction ofproteinase 6 days and 12 days of larval feeding correlated with levels of inhibitors in tomato leaves. Transgenic tomato plants, trans- formed with a cauliflower mosaic virus 35S-prosystemin inhibitor I and H protein levels. These experiments demon- cDNA in the antisense orientation that exhibited very low strate that resiLstnce of plants toward an insect pest can be systemic inducibility ofthe proteinase inhibitor I and II genes modulated by genetically engineering a gene encoding a com- (6), were employed to assess their effects on growth of ponent of the inducible systemic signaling system regulating a Manduca sexta larvae and for alterations in proteinase in- plant defensive response. hibitor synthesis in response to attacks by the larvae. Systemin, an 18-aa polypeptide isolated from tomato leaves, has been shown recently to be a powerful inducer of pro- MATERIALS AND METHODS teinase inhibitorprotein synthesis in tomato and plants Prosystemin Antisense Gene. Tomato plants (Lycopersicon (1). The properties of systemin strongly support a role for the esculentum, cv. Better Boy hybrid VFN) were transformed polypeptide as a systemic wound signal. When supplied to with a prosystemin antisense gene (6) composed of 747 bp of young tomato plants through their cut stems at femtomole the prosystemin cDNA (6) in the antisense orientation under levels, systemin induced de novo synthesis of proteinase the control of the constitutive cauliflower mosaic virus 35S inhibitor proteins. Moreover, 14C-labeled systemin placed in promoter and terminated with the 3' region of the T7 gene wounds was shown to be mobile, traveling systemically from from the Ti plasmid from Agrobacterium (9). The plasmid a wounded leaf to the upper leaves of treated tomato plants was introduced into Agrobacterium tumefaciens LBA4404, at about the same velocity as the endogenous wound signal which was employed to transform tomato plants. (1, 2). Radiolabeled systemin placed on wounds on tomato Tomato Transformation. Small aliquots ofA. tumefaciens, leaves was identified in exudates obtained from the transformed with the 35S-prosystemin cDNA antisense gene cut petioles oftreated leaves within an hour ofapplication (1). construct, were grown overnight in YEP medium containing These data are compatible with results obtained in poplar yeast extract (10 g/liter), Bacto Peptone (10 g/liter), NaCl (5 trees where the systemic wound signal also travels through g/liter), acetosyringone (3',5'-dimethoxy-4'-hydroxy-aceto- the to activate proteinase inhibitor synthesis (3). phenone; Aldrich) (11 mg/liter), tetracycline (3 mg/liter), and phloem kanamycin (10 mg/liter). Overnight cultures were diluted Thus these results contrast with recent reports suggesting with liquid MS medium (10) to 5 x 108 cells per ml of that electrical (4) or hydraulic (5) impulses are primary Agrobacterium for the infection of plant tissues (cocultiva- systemic signals that do not move through the phloem. tion). Isolation ofa cDNA and gene coding for systemin revealed Cotyledons isolated from germinated 10-day-old tomato that systemin is derived from the C-terminal region ofa larger seedlings were preconditioned by incubating them for 2 days precursor protein of 200 aa (6), called prosystemin. There- on tobacco feeder plates consisting of 2-day-old NT-1 to- fore, the active polypeptide must be released by proteolytic bacco suspension-cell cultures (11) plated on semi-solid MS cleavage by unidentified processing enzymes. The expres- medium containing 3% (wt/vol) sucrose, thiamine (1 mg/ sion of a gene containing the antisense prosystemin cDNA, liter), m-inositol (100 mg/liter), and 2,4-dichlorophenoxyace- regulated by the cauliflower mosaic virus 35S promoter in tic acid (0.2 mg/liter) (12). The cotyledons were wounded transgenic tomato plants, substantially abolished the sys- with a sterile syringe needle, cocultivated (immersed) for 30 temic wound induction of proteinase inhibitors in leaves (6). *Present address: Unidad de Investigacion en Biotechnologia Agri- The publication costs ofthis article were defrayed in part by page charge cola, Corporacion Colombiana de Investegacion Agropecuaria, payment. This article must therefore be hereby marked "advertisement" Apardo Aereo 151123, El Dorado, Bogota, D.C., Columbia. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8273 Downloaded by guest on September 29, 2021 8274 Plant Biology: Orozco-Cardenas et al. Proc. Natl. Acad. Sci. USA 90 (1993) min with the diluted culture of Agrobacterium, blotted with prosystemin gene (6) provided strong evidence that prosys- sterile filter paper, and incubated for 2 days in the feeder temin is required for wound-inducible synthesis ofproteinase plates. Thereafter, the explants were washed three times with inhibitors. Such plants provided an opportunity to test liquid MS medium. The last rinse contained cefotaxime (500 whether the suppressed wound inducibility of this defensive mg/liter). The tissues were blotted with sterile filter paper system would decrease resistance against larvae of the lep- and planted in selective medium containing the MS salts, B5 idopteran predator, M. sexta, which had previously been vitamins (13), myo-inositol (100 mg/liter), 3% sucrose, ade- shown to exhibit impaired growth when feeding on transgenic nine (40 mg/liter), Mes (0.5 g/liter), benzylaminopurine (2.5 tobacco plants expressing foreign tomato and potato inhibitor mg/liter), indole-3-acetic acid (1.0 mg/liter), cefotaxime (250 I and II genes (12, 16). mg/liter), carbenicillin (500 mg/liter), kanamycin (100 mg/ Ten newly hatched M. sexta larvae (=0.8 cm long and 4.0 liter), and phytoagar (8 g/liter). After 4 weeks of callus mg) were placed on leaves of tomato plants (418 inches high; growth, calli were transferred to the same selective medium containing only zeatin (2 mg/liter). Rooting of shoots was 1 inch = 2.54 cm) to initiate the experiments. M. sexta larval achieved in a medium containing indole-3-acetic acid (0.05 weights 10 and 14 days after the beginning of the feeding trial mg/liter) as the only hormone. The plantlets were then are shown in Fig. 1. Average weights of larvae after 14 days transferred to potting soil. of feeding on control nontransformed tomato plants were Three weeks after the transformed plants were transferred about one-third of larvae fed on the transgenic antisense to soil, the lower leaves on each small plant (plants were =20 tomato plants (Figs. 1 and 2). Furthermore, the larvae cm in length having about five developing leaves) were consumed much more foliage from the control plants than extensively wounded, and the amounts of wound-inducible larvae feeding on transgenic plants (Fig. 3). These data proteinase inhibitors I and II were determined immunologi- indicate that the expression of the antisense prosystemin cally in the expressedjuice ofupper leaves 24 h later by using gene has severely compromised the natural defense of the radial immunodiffusion assays in agar gels (14, 15). tomato plants and has made their leaves a much better food Primary transformants were grown to mature plants and source for the M. sexta larvae than leaves ofwild-type plants. selfed, and the progeny were analyzed for their abilities to The antinutritional effects of tomato and potato proteinase accumulate inhibitor I and II in response to wounding. One inhibitor proteins against several lepidopteran larvae have antisense line was selected that responded very weakly to been well documented (12, 16, 19, 20). The inhibitors have wounding in systemically inducing proteinase inhibitors syn- been shown to directly interfere with the activity of digestive thesis. This line showed a simple Mendelian segregation for enzymes in the insect guts, reducing their ability to digest only one copy of the antisense gene. Only homozygous their food (19, 21). Decreasing the ability of tomato plants to transgenic plants expressing the antisense gene were selected systemically synthesize the inhibitors has apparently re- for these experiments (6). moved a significant stress on the insects and allowed them to Manduca sexta Larvae Feeding Studies. Tobacco hornworm consume the antisense leaves at a much higher rate than (M. sexta) eggs (obtained from Carolina Biological Supply) control leaves to achieve increased growth rates. were sterilized and incubated as described (16). Newly McGurl et al. (6) had shown that a low constitutive level of hatched larvae were provided with an artificial food diet (17), prosystemin mRNA was present in tomato leaves and that the and first-instar larvae that were vigorous and uniformly sized A (=10 mm long) were selected within 3 days for feeding trials. levels increased in response to a systemic wound signal. Ten M. sexta larvae were placed directly on leaves of six basal level of prosystemin is apparently necessary for the 8-week-old control and six transgenic tomato plants. The plant to initiate the cascade of events that activate the plants were maintained in a growth chamber under 16 h of expression of the defensive proteinase inhibitor genes in daylight (300 ,uE m-2-s-1) at 30°C and 8 h of dark at 25°C. response to insect attacks. Northern blot analyses of total Proteinase inhibitor I and II concentrations were recorded RNA extracts obtained from leaves of control and transgenic at various times before and during the feeding trial by using tomato plants before insect damage and after 6 and 12 days immunological radial diffusion assays of leafjuice (14, 15). of feeding by M. sexta larvae are shown in Fig. 4. In control The effect of the expression of the antisense prosystemin plants, the levels of prosystemin mRNA increased substan- gene on larval growth was estimated by recording the final tially by day 6 in response to wounding by the insects. After larval weight and also observing the amount of leaf damage 12 days, the prosystemin mRNA level was substantially on the plants at the end of the feeding trial. higher than the initial level. In the transgenic tomato plants, Inhibitor I and II mRNA levels were determined in total RNA fractions, extracted from random leaves of wild-type and transgenic tomato plants before the feeding trial and 6- every 2 days during the feeding trial. Leaves were frozen in liquid nitrogen, ground to a fine powder, and immediately phenol/chloroform-extracted, and the nucleic acids were =S,~~~~~~~~~~~~~~~~~~~~~~~...... ethanol-precipitated, as described (12). The final yield oftotal._4Z A .... RNA was 150-400 p,g/100 mg of leaf tissue. Equal amounts ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ oftotal RNA were electrophoresed in agarose gels containing 1.4% formaldehyde and transferred to nitrocellulose filters, as described by Maniatis et al. (18). Filters were hybridized ~~~~~~~~~~~~~~~~~~~~~~~~~~...... o::.::.:. to nick-translated 32P-labeled cDNA fragments, isolated after digestion of the prosystemin cDNA clone with Bgl II and BamHI (6). After washing under low-stringency conditions -~~~~~~~~~~~~~~~~~~..{) ...... (6), the filters were exposed to film at -70°C to detect the 14 days 10 days 14 days prosystemin mRNA in the control (wild type) plants and/or Wild type Antisense the transgenic tomato. the prosystemin antisense mRNA in FIG. 1. Growth of Manduca sexta larvae feeding on leaves of control and transgenic tomato plants expressing a prosystemin RESULTS AND DISCUSSION antisense gene. Ten first-instar larvae (0.8 cm long) were placed on six control plants and six transgenic plants. Values are the average The severely decreased wound inducibility ofinhibitors I and of three feeding experiments. Error bars indicate the standard II in tomato plants constitutively expressing an antisense deviation of the error. Downloaded by guest on September 29, 2021 Plant Biology: Orozco-Cardenas et al. Proc. Natl. Acad. Sci. USA 90 (1993) 8275

Control Antisense

0 6 12 0 6 12 Days FIG. 4. Northern blot analysis of total RNA extracts prepared FIG. 2. Manduca sexta larvae after feeding 14 days on the control from leaves of control nontransformed and transgenic antisense tomato plants (Upper) and transgenic antisense tomato plants (Low- tomato plants at 0 time and after 6 and 12 days offeeding by Manduca er). sexta larvae. Total RNA (10 pg) was prepared from leaves at each time point, separated by electrophoresis, blotted onto nitrocellulose, prosystemin mRNA did not show an increase at 6 days, but and probed with a nick-translated 32P-labeled prosystemin cDNA. after 12 days, the level had increased but was estimated to be only about one-third of the level found in the control tomato antisense plants increased but were 2-3 times lower than in the plants. control plants throughout this period (Fig. 5). The lower levels of prosystemin mRNA found in the The results ofthis study demonstrate that the manipulation transgenic antisense plants are likely the result ofthe inability of a gene coding for a component of an inducible signaling of the prosystemin gene to be induced by wounding, similar system can affect plant resistance toward insects. It is to the reason for the lower levels of proteinase inhibitors in possible that prosystemin may be regulating the synthesis of the leaves of wounded antisense plants. This result suggests other wound-inducible plant defense genes besides the pro- that pro.ystemin, and perhaps systemin itself, is involved teinase inhibitors. The amino acid sequence of prosystemin with the signaling of both proteinase inhibitors and prosys- has several repetitive sequence elements that are unrelated to temin. It took >6 days of constant insect attacks on the the single systemin sequence near the C terminus (22). It is transgenic plants to overcome the effects of the antisense possible, as has been found with polypeptide prohormones in mRNA, and only then could they begin to produce prosys- yeast and animal systems (23, 24), that prosystemin may be temin mRNA. proteolytically processed to produce biologically active poly- Fig. 5 shows the accumulation ofproteinase inhibitor I and other than systemin that may activate defensive II proteins in leafjuice obtained from control and transgenic responses or other responses related to wounding such as tomato leaves during the course of the feeding trials. After 2 wound healing. The inhibition of prosystemin production in days of feeding by M. sexta larvae on control tomato plant the transgenic antisense plants may, therefore, be suppress- leaves, the rates of proteinase inhibitor I and II proteins ing responses other thanjust proteinase inhibitor synthesis to remained almost linear until about the 8th day and then the make the plants a better food for the insect larvae. rates decreased. In leaves of transgenic tomato, inhibitors I In contrast to the data in this report, the overexpression of and II began to accumulate only after the 6th day ofthe feeding the prosystemin gene in the correct-sense orientation and experiment, corresponding to the beginning of the increased accumulation ofprosystemin mRNA transcripts, as observed by Northern blot analysis (Fig. 4). From 6 days to 14 days of larval feeding, the levels of inhibitor proteins in leaves of the 300

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Time, days FIG. 5. Accumulation ofinhibitor I and II proteins in undamaged leaves of untransformed control and transgenic tomato plants in response to feeding by Manduca sexta larvae. Inhibitor I, untrans- formed control plants (-*-); inhibitor I, antisense plants (- - m - -); FIG. 3. Control (Right) and transgenic antisense (Left) tomato inhibitor II, untransformed control plants (--o-); inhibitor II, plants after 14 days offeeding by Manduca sexta larvae (10 larvae per antisense plants (- - o- -). Leaf juice was assayed by the radial plant). immunodiffusion assay. Downloaded by guest on September 29, 2021 8276 Plant Biology: Orozco-Cardenas et al. Proc. Natl. Acad Sci. USA 90 (1993) under the control of a strong tissue-specific constitutive 7. Farmer, E. E. & Ryan, C. A. (1990) Proc. Natl. Acad. Sci. to regulate the expres- USA 87, 7713-7716. promoter might be a viable approach 8. Farmer, E. E. & Ryan, C. A. (1992) 4, 129-134. sion of proteinase inhibitor genes, or other defensive genes, 9. An, G., Ebert, P. R., Mitra, A. & Ha, S. B. (1988) in Plant to enhance plant resistance in crop plants against insect pests Molecular Biology Manual, eds. Gelvin, S. B., Schilperoort, and/or pathogens. On the other hand, the antisense technol- R. A. & Verma, D. P. S. (Kluwer, Dordrecht, The Nether- ogy used in this research might be used in the future to lands), pp. 1-19. the quality and nutritional value of plants. By 10. Murishuge, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-495. increase 11. An, G. (1985) Plant Physiol. 79, 568-570. inhibiting the synthesis and accumulation of toxic or antinu- 12. Narvaez-Vasquez, J., Orozco-Cardenas, M. L. & Ryan, C. A. tritional compounds that are induced by pest attacks, it may (1992) Plant Mol. Biol. 20, 1149-1157. be possible to widen the menu of plants in the diets of 13. Gamborg, 0. L., Miller, R. A. & Ojima, K. (1968) Exp. Cell domestic animals or, perhaps, even humans. Res. 50, 151-158. 14. Ryan, C. A. (1987) Anal. Biochem. 19, 434-440. We thank Dr. Javier Narvaez-Vasques for technical advice, Greg 15. Trautman, R., Cowan, K. M. & Wagner, G. G. (1971) Immu- Pearce for technical assistance and advice, and Greg Wichelns for nocytochemistry 8, 901-916. 16. Johnson, R., Narvaez, J., An, G. & Ryan, C. A. (1989) Proc. growing the plants used in this research. This research was supported Natl. Acad. Sci. USA 86, 9871-9875. in part by the Washington State University College of Agriculture 17. Bell, R. & Joachim, F. (1976) Ann. Entomol. Soc. Am. 69, and Home Economics Project 1791 and National Science Foundation 365-373. Grants DCB-9104542 and DCB-9117795. 18. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. 1. Pearce, G., Strydom, D., Johnson, S. & Ryan, C. A. (1991) Press, Plainview, NY). Science 253, 895-898. 19. Broadway, R. M. & Duffey, S. S. (1986) Insect Physiol. 32, 2. Nelson, C. E., Walker-Simmons, M., Makus, D., Zuroske, G., 827-833. Graham, J. & Ryan, C. A. (1983) in PlantResistance to Insects, 20. Broadway, R. M., Duffey, S. S., Pearce, G. & Ryan, C. A. ed. Hedin, P. A. (ACS, Washington, DC), pp. 103-122. (1986) Entomol. Exp. Appl. 41, 33-38. 3. Davis, J. M., Gordon, M. P. & Smit, B. A. (1991) Proc. Natl. 21. Applebaum, S. W. (1985) in Comprehensive Insect Physiology, Acad. Sci. USA 88, 2393-23%. Biochemistry and Pharmacology, eds. Kerkut, G. A. & Gil- 4. Wildon, D. C., Thain, J. F., Minchin, P. E. H., Gubb, I. R., bert, L. I. (Pergamon, New York), Vol. 4, pp. 279-311. Reilly, A. J., Skipper, Y. D., Doherty, H. M., O'Donnell, P. J. 22. McGurl, B. & Ryan, C. A. (1992) Plant Mol. Biol. 20, 405-409. & Bowles, D. J. (1992) Nature (London) 360, 62-65. 23. Wallis, M., Howell, S. L. & Taylor, K. W. (1985) The Bio- 5. Malone, M. (1992) Planta 187, 505-510. chemistry of the Polypeptide Hormones (Wiley, New York). 6. McGurl, B., Pearce, G., Orozco-Cardenas, M. L. & Ryan, 24. Polak, J. M., ed. (1989) Regulatory Peptides (Birkhaeuser, C. A. (1992) Science 2S5, 1570-1573. Berlin). Downloaded by guest on September 29, 2021