ENHANCED QUANTITATIVE RESISTANCE AGAINST FUNGAL DISEASE BY COMBINATORIAL EXPRESSION OF DIFFERENT BARLEY ANTIFUNGAL PROTEINS IN TRANSGENIC TOBACCO

cDNAs encoding three proteins from barley (Hordeum vulgare), a class-I I chitinase (CHI), a class-II beta-1,3-glucanase (GLU) and a Type-I ri bosome-inactivating protein (RIP) were expressed in tobacco plants und er the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern an d Western blot analysis. The leader peptides in CHI and GLU led to acc umulation of these proteins in the intercellular space of tobacco leav es. RIP, which is naturally deposited in the cytosol of barley endospe rm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays reveale d that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditiona l breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and prote ction against fungal attack resulting from their co-expression in plan ta was evaluated. Transgenic tobacco lines were generated with tandeml y arranged genes coding for RIP and CHI as well as GLU and CHI. The pe rformance of tobacco plants co-expressing the barley transgenes GLU/CH I or CHI/RIP in a Rhizoctonia solani infection assay revealed signific antly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressi ng a single barley transgene to a similar level. The data indicate syn ergistic protective interaction of the co-expressed antifungal protein s in vivo.