EXPRESSION OF A DEFENSE-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 a n enhanced CaMV 35S promoter. Progeny from three independent transform ants were analyzed genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley per oxidase is expressed and sorted to the intercellular space in the tran sgenic tobacco plants. The peroxidase can be extracted from the interc ellular space in two molecular forms from both barley and transgenic t obacco. The tobacco expressed forms are indistinguishable from the bar ley 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 cleav age site deduced from the cDNA sequence. Phenotype comparisons show th at the constitutive expression of Prx8 lead to growth retardation. How ever, an infection assay with the tobacco powdery mildew pathogen Erys iphe cichoracearum did not indicate that the transgenic plants had ach ieved enhanced resistance. (C) 1997 Elsevier Science Ireland Ltd.