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The expression of chloramphenical acetyl transferase (CAT) protein driven by the wound-inducible promoter from the proteinase inhibitor 11 K (pin2) gene was examined in whole tobacco (Nicotiana tabacum L.) plants under field conditions. Mechanical wounding of the field-grown leaves caused an accumulation of CAT protein in these leaves which begins several hours after wounding and continues to accumulate for about 36 hours. When sections of leaves were assayed for accumulation of CAT protein following wounding, the CAT protein was found to accumulate in the apical portions of the leaves. When endogenous insects attacked the leaves of transgenic plants grown in the field, the plants responded by inducing CAT protein. The mesophyll cells of the leaf were the site of expression of the CAT protein rather than the mid-vein or major veins within the leaf blade, indicating that the wound-inducible pin2 promoter specifically directs the synthesis of novel genes in tissues preferentially consumed by larval insects.are then free to act on their substrates, the plants' cell walls, to release small mol wt oligosaccharides (1,2,16). These oligosaccharides have been termed PIIF2. They are believed to be the signal which interacts with unwounded cells in an as yet undetermined manner to induce the de novo synthesis of proteinase inhibitor mRNA and protein (9). These genes have been used in whole plants to express the native inhibitors (17) or foreign proteins (18) One particular biochemical defense system utilized by plants against insects is directed against the digestive enzymes located in the insect gut. This system of proteinase inhibitors occurs in the seeds of almost all plant species ( 11) and in the foliage of solanaceous (potatoes and tomatoes) (10, 14) and leguminous (alfalfa) (6) plants.In response to insect attack, tomatoes and potatoes activate quiescent genes, which leads to the accumulation of at least two small protein families. The activation of these genes is complex and incompletely understood. Initially, wounding disrupts cells and specific enzymes, polygalacturonases, are released to the extracellular milieu. These polygalacturonases 'This work was supported by grants from the U.S. Department of Agriculture (87-CRCR-1-2518), the State of Iowa, and the Iowa Biotechnology Council. PlantsTobacco (Nicotiana tabacum L., cv Xanthi) plants transformed with a chimeric pin2-CA T construction were previously described (18). One line, Tr25, showed high levels of wound-inducible CAT activity and was chosen for further analysis. This line was made homozygous by self-fertilizing the plants. Selfed progeny in the R1 and R2 generations of Tr25 were grown on Murashige-Skoog (13) solid medium containing 200 gg/ml kanamycin. Since the pin2-CA T gene fusion is physically linked to a dominant kanamycin selectable marker (only 2.7 kb separates these genes), a test for a kanamycin resistance provides a good measure of the presence of the linked pin2-CA T gene. The R1 progeny segregated in a 3:1 ratio, indicating that the...

A hybrid poplar clone has been transformed with a previously constructed wound-inducible potato proteinase inhibitor (pin2) – chloramphenicol acetyltransferase (CAT) chimeric gene linked to a nopaline synthase (nos) – neomycin phosphotransferase II (NPT II) selectable marker gene. The Populusalba × Populusgrandidentata Hansen clone was transformed by means of leaf cocultivation with Agrobacteriumtumefaciens strain A281 containing a binary vector. Shoots were regenerated and rooted on selective medium containing kanamycin sulfate. Subsequently, plants were established in soil for greenhouse and field growth. NPT II activity from the nos – NPT II selectable marker gene was observed in leaf extracts, thereby confirming expression of the transferred selectable marker gene in poplar. Southern hybridization confirmed the incorporation of a single copy of the pin2–CAT construction into the genome of the transformed hybrid poplars. Northern analysis of transgenic poplar leaves demonstrated that the potato pin2–CAT gene construction also was inducible in this woody dicotyledon. Thus, the wound-inducible promoter from an herbaceous dicot functions in a distinct family of woody dicots.

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