Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity.
BACKGROUND
Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors.


RESULTS
A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal membranes.


CONCLUSIONS
A berry specific cDNA clone, Vv-AMP1, was isolated and characterized and shown to encode a plant defensin. Recombinant Vv-AMP1 displayed non-morphogenic antifungal activity against a broad spectrum of fungi, probably altering the membrane permeability of the fungal pathogens. The expression of this peptide is highly regulated in Vitis vinifera, hinting at an important defense role during berry-ripening.
de Beer
Abré
A
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Faculty of AgriSciences, Stellenbosch University, Stellenbosch 7600, South Africa. abre@sun.ac.za
Vivier
Melané A
MA
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D023082:Defensins / Q000235:genetics* / Q000302:isolation & purification; D005638:Fruit / Q000378:metabolism*; D005659:Fungicides, Industrial / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein; D027843:Vitis / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D018744:DNA, Plant; D023082:Defensins; D005659:Fungicides, Industrial; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins