5.3.11
ISOLATION AND CHARACTERIZATION OF NOVEL PLANT-ACTIVE PROMOTERS FROM BANANA BUNCHY TOP VIRUS

SR HERMANN, B DUGDALE, DK BECKER, RM HARDING and JL DALE

Centre for Molecular Biotechnology, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia

Background and objectives
Banana bunchy top disease, caused by banana bunchy top virus (BBTV), is considered the most serious viral disease of bananas worldwide. BBTV has a multicomponent, circular ssDNA genome, and at least six ssDNA components (BBTV DNA 1-6) have been identified in all infections tested [1]. Each component is approximately 1 kb in size and contains one major ORF in the viron sense. Recently, seven additional components, all potentially encoding replication proteins (as does BBTV DNA 1), have been found in a number of South-East Asian bunchy top isolates. As these components do not appear to be present in all infections, they may not be necessary for infection and may represent DNA satellites of BBTV. We have isolated two of these new components, BBTV-S1 and S2, from a Taiwanese isolate of BBTV, and have studied the promoter activity associated with the intergenic regions of these components to determine the tissue specificity of gene expression and the capability of these promoters to express potential virus resistance genes in transgenic plants.

Results and conclusions
Potential promoter (intergenic) regions of BBTV components S1 and S2 have been introduced into constructs to drive the uidA reporter gene (encoding GUS). The activities of these promoters have been assayed in both tobacco and banana (transiently and stably). In transient assays with tobacco callus, GUS activities from constructs incorporating the S1 and S2 promoters were comparable to that of uidA constructs driven by the CaMV 35S promoter. In transgenic tobacco plants, the promoters showed different patterns of expression. The S1 promoter (intergenic region of component S1) expressed weakly in root vascular tissue, with little or no detectable activity in leaves and flowers. Interestingly, a larger promoter region including 300 bp of the S1 ORF, 5 to the S1 promoter, significantly enhanced expression in the vascular tissue of both roots and leaves. This indicates the presence of one or more cis-acting or enhancer elements within the ORF of S1. The S2 promoter expressed strongly in root hairs and meristems, sepal trichomes and in pollen. Expression in vascular tissue of the leaves and roots was weak. Transient expression of both the S1 and S2 promoter regions driving uidA was very low, and sometimes undetectable, in a banana embryogenic cell suspension. However, the inclusion of the ubiquitin intron 5' of the uidA gene increased expression 10-fold. Stably transformed transgenic bananas are currently being regenerated. This study indicates that promoters derived from BBTV components S1 and S2 are promising candidates to drive resistance transgenes or selectable markers in transgenic bananas.

References
1. Burns T, Harding R, Dale J, 1995. Journal of General Virology 76, 1471-1482.