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Expression Ofan Antisense Prosystemin Gene in Tomato Plants

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Expression Ofan Antisense Prosystemin Gene in Tomato Plants Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8273-8276, September 1993 Plant Biology Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae (protelnae inhibitors/plant defense/trausgenic tomato plants/signal transduction) 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 insects 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 cytoplasm, which is then to 100-125 pg/g of leaf tissue after 14 days. In control plants, converted to the powerful signaling molecule jasmonic acid. 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 transcription 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 insect 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 potato 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 phloem 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).
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