1.3.35
CHARACTERIZATION OF THE PROMOTER OF AN APPLE IPR-PROTEIN GENE IN TRANSGENIC TOBACCO

H POHRINGER1, D MOLL1, K HOFFMANN-SOMMERGRUBER2, B WATILLON3, H KATINGER1 and M AIMER DA CAMARA MACHADO1

1Institute of Applied Microbiology, University of Agricultural Sciences, 1190 Vienna, Austria; 2Institute of General and Experimental Pathology, University of Vienna, 1090 Vienna, Austria; 3Unite de Biologie Moleculaire et Physiologie Animale, Faculte des Sciences Agronomique de Gembloux, 5030 Gembloux, Belgium.

Background and objectives
Analysis of the deduced amino-acid sequence of Mat dl cDNA [1], coding for the 18 kDa major apple allergen, revealed high homology to intracellular pathogen-related proteins (IPR-proteins) found in various other plants. Members of this ubiquitous class of proteins show high similarity, are encoded by multiple genes and have been shown to be transcriptionally unregulated in response to microbial attack, fungal elicitors, wounding and stress. However, the biological function of Mat dl is still unclear and, up to now, induction upon pathogen challenge has not been proved. To elucidate the gene expression pattern of Mat dl we identified and investigated its 5' regulating sequences in terms of inducibility and tissue and/or organ specifity.

Material and methods
The complete coding region of the Mat dl gene and 1362 bp of the 5' upstream region were isolated from a genomic library of Malus domestica cv. McIntosh 'Wijcik' and fully sequenced. We constructed a promoter-glucuronidase (GUS) fusion by cloning the available promoter length (1.3 kb), the 5' non-translated region and five codons of Mat dl in-frame to the reporter gene. This full-length construct, various promoter deletion mutants and a control construct harbouring the constitutive CaMV 35S promoter were transformed into tobacco, and several independent transgenic lines were regenerated.

Results and conclusions
The encoded amino-acid sequence of the genomic Mat dl clone showed 87.4% identity to Mat dl cDNA and 99.4% to pAP15 cDNA [2], a recently identified isoform of the Mat dl in cv. Golden Delicious. The coding region (480 bp) is interrupted by an intron spanning 170 bp at amino-acid position 62, which is highly conserved among IPR proteins. However, no consensus motive described for promoters of PR genes matched unambigously with the promoter sequence of the Mat dl gene. Nevertheless the 1.3 kb promoter length proved to be sufficient for GUS expression in transgenic tobacco. Histochemical GUS staining in in vitro plants showed a strong promoter activity of the full-length construct, comparable to the 35S promoter.

However, analysis of GUS expression of greenhouse-acclimatized plants showed a development-dependent promoter activity, which increased from younger to older leaves, irrespective of the age of the plants. After challenge infection with pathogenic viruses (tobacco mosaic virus, tobacco etch virus and tobacco vein mottling virus), GUS activity was dramatically enhanced, particularly in young leaves. This induction seemed not to be pathogen-specific, as it could also be obtained with fungal elicitors (Botrytis cinerea) as well as reduced glutathione.

References
1. Vanek-Krebitz M, Hoffmann-Sommergruber K, Laimer da Camara Machado M, et al., 1995. Biochemical and Biophysical Research Communications 214, 538-551.
2. Atkinson RG, Perry J, Matsui T et al., 1996. New Zealand Journal of Crop Horticultural Science 24, 103-107.