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Published on March 7, 2002; 10.1104/pp.010794

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Received August 3, 2001
Returned for revision October 31, 2001
Accepted December 21, 2001

Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins

Isabelle E.J.A. François , Miguel F.C. De Bolle , Geoff Dwyer , Inge J.W.M Goderis , Piet F.J. Woutors , Peter D. Verhaert , Paul Proost , Wim M.M. Schaaper , Bruno P.A. Cammue *, and Willem F. Broekaert

Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B--3001 Heverlee, Belgium (I.E.J.A.F., M.F.C.D.B., G.D., I.J.W.M.G., P.F.J.W., B.P.A.C., W.F.B.); Flanders Interuniversity Institute of Biotechnology, Rijvisschestraat 120, B--9052 Gent, Belgium (M.F.C.D.B., I.J.W.M.G., P.F.J.W., B.P.A.C.); Afdeling Vergelijkende Fysiologie en Morfologie Dieren, Katholieke Universiteit Leuven, Naamsestraat 59, B--3000 Leuven, Belgium (P.D.V.); Departement Microbiologie en Immunologie, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B--3000 Leuven, Belgium (P.P.); and Institute for Animal Science and Health (ID-DLO), P.O. Box 65, Lelystad 8200 AB, The Netherlands (W.M.M.S.)

* Corresponding author; email: Bruno.Cammue{at}agr.kuleuven.ac.be.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.






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