Transposable elements have been used effectively as tools for gene tagging in plants such as maize and snapdragon, where endogenous transposable elements have been detected and characterized. Such elements have not been detected in tomato, although a segment of DNA having the appearance of a transposable element has been found in a spacer region of the tomato Cab-1 gene cluster (Bernatzky et al. 1988). The discovery by Barbara Baker and her colleagues that the well characterized Ac element of maize would function in tobacco (Baker et al. 1986) raised the possibility that this element would also function in tomato. This was subsequently shown to be the case by Yoder et al. (1988).
A transposable element by itself has no detectable phenotype, therefore it is essential that the element be located in a reporter gene that can be monitored. Previous studies of Ac activity in dicotyledonous plants have detected transposition by Southern analysis (Baker et al. 1986, Yoder et al. 1988) or through the activation of a gene encoding neomycin phosphotransferase (Baker et al, 1987). Expression of this gene after transposition confers kanamycin resistance to cells carrying the active gene. Unfortunately, neither of these methods permits the direct visualization of sectors in which transpositions have occurred. This report describes a transposable element/reporter gene combination based on the Beta-glucuronidase (GUS) gene (Jefferson et al. 1987) Sectors of tissue that are expressing the GUS gene can be readily identified using a histochemical stain based on the compound X-gluc (5-bromo-4-chloro-3-indoyl glucuronide), which forms a blue precipitate when cleaved by the GUS enzyme.
The transposable element used in this construction was an Ac element recovered from the Pvv allele of maize by Dr Tom Peterson of the Cold Spring Harbor Laboratories. Bgl II linkers were placed approximately 25 bp from each end of Ac, and the element was inserted at a BamH I site located between the Cauliflower Mosaic Virus 35S promoter and GUS coding region of the plasmid pBI121 (Jefferson et al. 1987). The Ac/GUS combination on this plasmid is located on the T-DNA region of the Agrobacterium binary vector Bin 19, which also carries a kanamycin resistance selectable marker. The plasmid was transferred by triparental mating into Agrobacterium tumefaciens strain LBA4404 and used to infect both tobacco leaf and tomato (cv. VFN-8) cotyledon pieces by the method of Fillatti et al. (1987). Kanamycin resistant plants were obtained and tested for expression of the GUS gene following Ac transposition.
Sectors of cells expressing the GUS gene have been detected in 19 out of 20 tobacco plants carrying the Ac/GUS construction. The sectors that have been detected thus far follow patterns expected for clonal lineages in leaf, petiole and root tissue. Preliminary Southern data indicate that the appearance of GUS expressing sectors is correlated with molecular evidence of transposition. Sectors of GUS-expressing cells were not observed in control transformants containing either a defective Ac element or a intact 35S-GUS gene. Similar experiments in tomato are less advanced, however sectors of GUS-expressing tissue have been observed using the X-gluc stain on calli and shoots containing the Ac-GUS construction and it is anticipated that results similar to those observed in tobacco will be obtained.
Literature cited:
Baker, B., Schell, J., Lorz, H. and Fedoroff, N. (1986) Transposition of the maize controlling element "Activator" in tobacco. Proc. Natl. Acad. Sci. USA 83: 4844-4848
Baker, B., Coupland, G., Fedoroff, N., Starlinger, P. and Schell, J. (1987) Phenotypic assay for excision of the maize controlling element "Activator" in tobacco. EMBO J. 6: 1547-1554
Bernatzky, R., Pichersky, E., Malik, V.S. and Tanksley, S.D. (1988) CR1- a dispersed repeated element associated with the Cab-1 locus in tomato. Plant Mol. Biol. 10: 423-433
Fillatti, J.J., Kiser, J., Rose, R. and Comai, L. (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. BioTechnology 5: 726-730.
Jefferson, R.A., Kavanaugh, T.A. and Bevan, M.W. (1987) GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907