Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

Tobacco plants (Nicotiana benthamiana L.) have been transformed with a T-DNA vector construct carrying the cDNA pBH6-301, encoding the major pathogen induced leaf peroxidase (Prx8) of barley, under control of an enhanced CaMV 35S promoter. Progeny from three independent transformants were analyzed genetically, phenotypically and biochemically.

The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting.

Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from transgenic tobacco was blocked by pyroglutamate, after the removal of which, N-terminal sequencing verified the transit signal-peptide cleavage site deduced from the cDNA sequence. Phenotype comparisons show that the constitutive expression of Prx8 lead to growth retardation.

However, an infection assay with the tobacco powdery mildew pathogen Erysiphe cichoracearum did not indicate that the transgenic plants had achieved enhanced resistance. (C) 1997 Elsevier Science Ireland Ltd.