BackgroundPlant defensins are an important component of the innate defence system of plants where they form protective antimicrobial barriers between tissue types of plant organs as well as around seeds. These peptides also have other activities that are important for agricultural applications as well as the medical sector. Amongst the numerous plant peptides isolated from a variety of plant species, a significant number of promising defensins have been isolated from Brassicaceae species. Here we report on the isolation and characterization of four defensins from Heliophila coronopifolia, a native South African Brassicaceae species.ResultsFour defensin genes (Hc-AFP1-4) were isolated with a homology based PCR strategy. Analysis of the deduced amino acid sequences showed that the peptides were 72% similar and grouped closest to defensins isolated from other Brassicaceae species. The Hc-AFP1 and 3 peptides shared high homology (94%) and formed a unique grouping in the Brassicaceae defensins, whereas Hc-AFP2 and 4 formed a second homology grouping with defensins from Arabidopsis and Raphanus. Homology modelling showed that the few amino acids that differed between the four peptides had an effect on the surface properties of the defensins, specifically in the alpha-helix and the loop connecting the second and third beta-strands. These areas are implicated in determining differential activities of defensins. Comparing the activities after recombinant production of the peptides, Hc-AFP2 and 4 had IC50 values of 5-20 μg ml-1 against two test pathogens, whereas Hc-AFP1 and 3 were less active. The activity against Botrytis cinerea was associated with membrane permeabilization, hyper-branching, biomass reduction and even lytic activity. In contrast, only Hc-AFP2 and 4 caused membrane permeabilization and severe hyper-branching against the wilting pathogen Fusarium solani, while Hc-AFP1 and 3 had a mild morphogenetic effect on the fungus, without any indication of membrane activity. The peptides have a tissue-specific expression pattern since differential gene expression was observed in the native host. Hc-AFP1 and 3 expressed in mature leaves, stems and flowers, whereas Hc-AFP2 and 4 exclusively expressed in seedpods and seeds.ConclusionsTwo novel Brassicaceae defensin sequences were isolated amongst a group of four defensin encoding genes from the indigenous South African plant H. coronopifolia. All four peptides were active against two test pathogens, but displayed differential activities and modes of action. The expression patterns of the peptide encoding genes suggest a role in protecting either vegetative or reproductive structures in the native host against pathogen attack, or roles in unknown developmental and physiological processes in these tissues, as was shown with other defensins.

The tyrocidines, a complex of analogous cyclic decapeptides produced by Bacillus aneurinolyticus, exhibited noteworthy activity against a range of phytopathogenic fungi, including Fusarium verticillioides, Fusarium solani and Botrytis cinerea. The activity of the tyrocidine peptide complex (Trc mixture) and purified tyrocidines exhibited minimum inhibition concentrations below 13 mg ml "1 (~10 mM) and was significantly more potent than that of the commercial imidazole fungicide, bifonazole. Although the tyrocidines' activity was negatively influenced by the presence of Ca 2+, it remained unaffected by the presence of Mg 2+ , Na + and K + . Microscopic analysis revealed significant impact on the morphology of F. solani and Bot. cinerea including retarded germination and hyperbranching of hyphae. Studies with membrane-impermeable dyes, SYTOX green and propidium iodide suggested that the main mode of action of tyrocidines involves the disruption of fungal membrane integrity. Because of the tyrocidines' broad spectrum and potent antifungal activity, possible multiple targets reducing the risk of overt resistance and general salt tolerance, they are promising candidates that warrant further investigation as bio-fungicides.

BackgroundLatest 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.ResultsA 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.ConclusionA 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.

Antibiotic production as a defence mechanism is a characteristic of a wide variety of organisms. In natural evolutionary adaptation, cellular events such as sporulation, biofilm formation and resistance to antibiotics enable some microorganisms to survive environmental and antibiotic stress conditions. The two antimicrobial cyclic peptides in this study, gramicidin S (GS) from Aneurinibacillus migulanus and the lipopeptide surfactin (Srf) from Bacillus subtilis, have been shown to affect both membrane and intercellular components of target organisms. Many functions, other than that of antimicrobial activity, have been assigned to Srf. We present evidence that an additional function may exist for Srf, namely that of a detoxifying agent that protects its producer from the lytic activity of GS. We observed that Srf producers were more resistant to GS and could be co-cultured with the GS producer. Furthermore, exogenous Srf antagonized the activity of GS against both Srf-producing and non-producing bacterial strains. A molecular interaction between the anionic Srf and the cationic GS was observed with circular dichroism and electrospray MS. Our results indicate that the formation of an inactive complex between GS and Srf supports resistance towards GS, with the anionic Srf forming a chemical barrier to protect its producer. This direct detoxification combined with the induction of protective stress responses in B. subtilis by Srf confers resistance toward GS from A. migulanus and allows survival in mixed cultures.