Chloramphenicol Acetyl Transferase (CAT) Protein Is Expressed in Transgenic Tobacco in Field Tests following Attack by Insects

Abstract

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...

Wounding Nicotiana tabacum Leaves Causes a Decline in Endogenous Indole-3-Acetic Acid

“…Eventually, the levels of IAA decline by two-to threefold in the 24 h following the wound. The kinetics of this decline in IAA levels is very similar to the kinetics of activation of the proteinase inhibitor gene system in either normal tomato plants (13), or in transgenic tobacco plants (28). This decline in IAA levels in wounded tobacco foliage is consistent with a role of IAA in the regulation of the inhibitor II gene system.…”

“…When tobacco plants bearing thepin2-CAT gene were examined for their expression of CAT protein, they expressed CAT protein in a manner which was identical with the wild-type expression of proteinase inhibitor II in young tomato plants (26,28). Therefore, these plants make a good model system for the study of the wound-inducible genes.…”

“…Both wild-type and transformed progeny plants were used. The transformed plants were previously described (28). They were an R4 homozygous line of transgenic plants (four self-pollinations after regeneration, RO) containing a pin2-CAT construction which was also previously described (27).…”

Cytokinin-mediated insect resistance in Nicotiana plants transformed with the ipt gene

“…The chimeric PI-II-ipt gene codes for the first enzyme in the cytokinin biosynthetic pathway and its expression is regulated by the promoter from the PI-IIK gene [19,30,36]. Fusions with the /%glucuronidase or chloramphenicol acetyl transferase (CAT) reporter genes indicated that the PI-IIK promoter directs gene expression in tissues preferentially consumed by insects and that CAT gene expression in leaves of transgenic tobacco plants had no effect on the extent of leaf consumption by various insects [20,35,36]. Ten kanamycin-resistant, independently transformated shoots were chosen and grown to maturity.…”

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

“…99,1992 inducible gene, we chose as our starting material a line of transgenic tobacco, Tr25, that contains a pin2-CAT construct (31). These plants show high levels of wound-inducible CAT activity from the transgene and have been well characterized (32).…”