Plant Physiol, April 2002, Vol. 128, pp. 1346-1358
Transgenic Expression in Arabidopsis of a Polyprotein Construct
Leading to Production of Two Different Antimicrobial
Proteins1
Isabelle E.J.A.
François,
Miguel F.C.
De Bolle,
Geoff
Dwyer,2
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.
Broekaert3
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.)
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.
1
This research was partially supported by the
"Instituut voor de aanmoediging van Innovatie door Wetenschap en
Technologie in Vlaanderen" (grant no. SB971156), by the European
Union (grant no. AIR2-CT94-1356), and by Zeneca Agrochemicals (UK).
I.E.J.A.F. is the recipient of a predoctoral fellowship of the
"Instituut voor de aanmoediging van Innovatie door Wetenschap en
Technologie in Vlaanderen."
2
Present address: Western Australia State Agriculture
Biotechnology Centre, Division of Science and Engineering, Murdoch
University, Perth, WA 6750, Australia.
3
Present address: CropDesign N.V., Technologiepark 3, B-9502 Gent, Belgium.
*
Corresponding author; e-mail
Bruno.Cammue{at}agr.kuleuven.ac.be; fax 32-16-32-19-66.
© 2002 American Society of Plant Physiologists
