Transgenic Expression in Arabidopsis of a Polyprotein Construct Leading to Production of Two Different Antimicrobial Proteins1

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 merckiiseeds and RsAFP2 originating from Raphanus sativusseeds, which are linked by an intervening sequence (“linker peptide”) originating from a natural polyprotein occurring in seed ofImpatiens 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]  W. Broekaert,et al.  A set of modular plant transformation vectors allowing flexible insertion of up to six expression units , 2002, Plant Molecular Biology.

[2]  R. Meagher,et al.  Phytodetoxification of hazardous organomercurials by genetically engineered plants , 2000, Nature Biotechnology.

[3]  G. Kishore,et al.  Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production , 1999, Nature Biotechnology.

[4]  J. Denecke,et al.  The Endoplasmic Reticulum—Gateway of the Secretory Pathway , 1999, Plant Cell.

[5]  C. Halpin,et al.  Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants. , 1999, The Plant journal : for cell and molecular biology.

[6]  M. Matzke,et al.  Position effects and epigenetic silencing of plant transgenes. , 1998, Current opinion in plant biology.

[7]  Henry Daniell,et al.  Containment of herbicide resistance through genetic engineering of the chloroplast genome , 1998, Nature Biotechnology.

[8]  M. McPherson,et al.  Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs , 1998, Planta.

[9]  I. J. Evans,et al.  A Novel Family of Small Cysteine-rich Antimicrobial Peptides from Seed of Impatiens balsamina Is Derived from a Single Precursor Protein* , 1997, The Journal of Biological Chemistry.

[10]  Franky R. G. Terras,et al.  Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. , 1996, The Plant cell.

[11]  M. Mann,et al.  Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry. , 1996, Current opinion in biotechnology.

[12]  Franky R. G. Terras,et al.  Plant Defensins: Novel Antimicrobial Peptides as Components of the Host Defense System , 1995, Plant physiology.

[13]  F. van Leuven,et al.  Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae , 1995, FEBS letters.

[14]  S. Farrand,et al.  Expression of Multiple Eukaryotic Genes from a Single Promoter in Nicotiana , 1995, Bio/Technology.

[15]  N V Raikhel,et al.  Small cysteine-rich antifungal proteins from radish: their role in host defense. , 1995, The Plant cell.

[16]  R. Dixon,et al.  Enhanced Protection Against Fungal Attack by Constitutive Co–expression of Chitinase and Glucanase Genes in Transgenic Tobacco , 1994, Bio/Technology.

[17]  G. Pont-Kingdon.  Construction of chimeric molecules by a two-step recombinant PCR method. , 1994, BioTechniques.

[18]  J. Marcos,et al.  In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide , 1994, Plant Molecular Biology.

[19]  B. Cornelissen,et al.  Virus and Fungal Resistance: From Laboratory to Field , 1993 .

[20]  E. Krebbers,et al.  An aspartic proteinase present in seeds cleaves Arabidopsis 2 S albumin precursors in vitro. , 1993, The Journal of biological chemistry.

[21]  J. Schell,et al.  New plant binary vectors with selectable markers located proximal to the left T-DNA border , 1992, Plant Molecular Biology.

[22]  V. Walbot,et al.  Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. , 1992, Nucleic acids research.

[23]  Franky R. G. Terras,et al.  Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. , 1992, The Journal of biological chemistry.

[24]  J. Vanderleyden,et al.  An automated quantitative assay for fungal growth inhibition , 1990 .

[25]  M. Bibb,et al.  A cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. , 1990, Nucleic acids research.

[26]  J. Carrington,et al.  A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Robert Kay,et al.  Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes , 1987, Science.

[28]  M. Chilton,et al.  Structure and transcription of the nopaline synthase gene region of T-DNA. , 1983, Nucleic acids research.

[29]  D. Dennis,et al.  Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L. , 1977, Plant physiology.

[30]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[31]  I. Mitsuhara,et al.  Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants. , 1998, Plant & cell physiology.

[32]  R. Jackson,et al.  Internal initiation of mRNA translation in eukaryotes. , 1994, Genetic engineering.

[33]  J. Ellis,et al.  In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants , 1993 .