Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium

Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol fungus Trichoderma harzianum. Calli from four primary trans formants had high levels of endochitinase activity, like the plants from which they were derived. Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions. Western blots demonstrated the presence of the expected 40-kDa T. harzianum protein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration was increased up to 34-fold by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions. probably due to the secretion and concentration of endogenous enzymes.