A gene that encodes systemin, a mobile 18-amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato leaves, has been isolated from tomato, Lycopersicon esculentum. Induction of proteinase inhibitors in plants is a response to insect or pathogen attacks. The gene has 10 introns and 11 exons, ten of which are organized as five homologous pairs with an unrelated sequence in the eleventh, encoding systemin. Systemin is proteolytically processed from a 200-amino acid precursor protein, prosystemin. Prosystemin messenger RNA was found in all organs of the plant except the roots and was systemically wound-inducible in leaves. Tomato plants transformed with an antisense prosystemin complementary DNA exhibited greatly suppressed systemic wound induction of proteinase Inhibitor I and II synthesis in leaves.

Tomato 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 ofthe 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. 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).

The growth rates of Manduca sexta (tobacco hornworm) larvae feeding on tomato plants constitutively expressing a prosystemin antisense gene were =3 times higher than growth rates of larvae feeding on nontransformed control plants. The levels of proteinase inhibitor I and inhibitor H proteins in leaves of tomato plants expressing the antisense prosystemin gene remained at undetectable levels until the sixth day of larval feeding and then increased throughout the plants to A model for the signaling of the inhibitor genes has been presented (7, 8) in which systemin is released, as the result of wounding by attacking insects or other mechanical damage to leaves, and is translocated throughout the plant where it interacts with receptors in the plasma membrane of both nearby and distal cells. It was proposed that the interaction with receptors activates a lipase, releasing membranederived linolenic acid into the cytoplasm, which is then converted to the powerful signaling molecule jasmonic acid. Jasmonic acid, or a derivative, is proposed to interact with transcription factors to activate the inhibitor genes (7,8).In this study, we investigate whether insect resistance can be affected by genetically modifying the function of a component of the signaling system for the induction of proteinase inhibitors in tomato leaves. Transgenic tomato plants, transformed with a cauliflower mosaic virus 35S-prosystemin cDNA in the antisense orientation that exhibited very low systemic inducibility of the proteinase inhibitor I and II genes (6), were employed to assess their effects on growth of Manduca sexta larvae and for alterations in proteinase inhibitor synthesis in response to attacks by the larvae. MATERIALS AND METHODSProsystemin Antisense Gene. Tomato plants (Lycopersicon esculentum, cv. Better Boy hybrid VFN) were transformed with a prosystemin antisense gene (6) composed of 747 bp of the prosystemin cDNA (6) in the antisense orientation under the control of the constitutive cauliflower mosaic virus 35S promoter and terminated with the 3' region of the T7 gene from the Ti plasmid from Agrobacterium (9). The plasmid was introduced into Agrobacterium tumefaciens LBA4404, which was employed to transform tomato plants.Tomato Transformation. Small aliquots ofA. tumefaciens, transformed with the 35S-prosystemin cDNA antisense gene construct, were grown overnight in YEP medium containing yeast extract (10 g/liter), Bacto Peptone (10 g/liter), NaCl (5 g/liter), acetosyringone (3',5'-dimethoxy-4'-hydroxy-acetophenone; Aldrich) (11 mg/liter), tetracycline (3 mg/liter), and kanamycin (10 mg/liter). Overnight cultures were diluted with liquid MS medium (10) to 5 x 108 cells per ml of Agrobacterium for the infection of plant tissues (cocultivation).Cotyledons isolated from germinated 10-day-old tomato seedlings were preconditioned by incubating them for 2 days on tobacco feeder plates consisting of 2-day-old NT-1 tobacco suspension-cell cultures (11) plated on semi-solid MS medium containing 3% (wt/vol) sucrose, thiamine (1 m...

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the bglucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-b-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, con®rmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Abbreviations: GUS = b-glucuronidase; MJ = methyl jasmonate; Prosys-GUS = prosystemin-b-glucuronidase; Correspondence to: C.A. Ryan; Fax: 1 (509) 335 7643; Tel: 1 (509) 335 3304

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.

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