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Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

ABSTRACT: Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an ?-helix and three anti-parallel ?-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved ?-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of ?2 and ?3 strands and the interposed loop. The ?-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their ?-core motifs. The MsDef1-?4 variant in which the ?-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the ?-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic ?-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-?4, MtDef4 and peptides derived from the ?-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the ?-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.

SUBMITTER: Sagaram US

PROVIDER: S-EPMC3076432 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

Sagaram Uma Shankar US Pandurangi Raghoottama R Kaur Jagdeep J Kaur Jagdeep J Smith Thomas J TJ Shah Dilip M DM

PloS one 20110413 4

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyph ...[more]

PMID: 21533249

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Project description:IntroductionFusarium graminearum is a most destructive fungal pathogen that causes Fusarium head blight (FHB) disease in cereal crops, resulting in severe yield loss and mycotoxin contamination in food and feed. Silver nanoparticles (AgNPs) are extensively applied in multiple fields due to their strong antimicrobial activity and are considered alternatives to fungicides. However, the antifungal mechanisms and the effects of AgNPs on mycotoxin production have not been well characterized.ObjectivesThis study aimed to investigate the antifungal activity and mechanisms of AgNPs against both fungicide-resistant and fungicide-sensitive F. graminearum strains, determine their effects on mycotoxin deoxynivalenol (DON) production, and evaluate the potential of AgNPs for FHB management in the field.MethodsScanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscopy were used to examine the fungal morphological changes caused by AgNPs. In addition, RNA-Seq, qRT-PCR, and western blotting were conducted to detect gene transcription and DON levels.ResultsAgNPs with a diameter of 2 nm exhibited effective antifungal activity against both fungicide-sensitive and fungicide-resistant strains of F. graminearum. Further studies showed that AgNP application could impair the development, cell structure, cellular energy utilization, and metabolism pathways of this fungus. RNA-Seq analysis and sensitivity determination revealed that AgNP treatment significantly induced the expression of azole-related ATP-binding cassette (ABC) transporters without compromising the control efficacy of azoles in F. graminearum. AgNP treatment stimulated the generation of reactive oxygen species (ROS), subsequently induced transcription of DON biosynthesis genes, toxisome formation, and mycotoxin production.ConclusionThis study revealed the underlying mechanisms of AgNPs against F. graminearum, determined their effects on DON production, and evaluated the potential of AgNPs for controlling fungicide-resistant F. graminearum strains. Together, our findings suggest that combinations of AgNPs with DON-reducing fungicides could be used for the management of FHB in the future.

Project description:Application of synthetic fungicides in agricultural commodities has been restricted due to development of fungicide resistance fungi and deleterious impact on environment and health of farm animals and humans. Hence, there is an urge for development of mycobiocides, and the present study was undertaken to determine the antifungal activity of Cymbopogon martinii essential oil (CMEO) on post-harvest pathogen Fusarium graminearum. The CMEO was extracted by hydrodistillation and GC-MS chemical profile revealed the presence of 46 compounds and abundant was geraniol (19.06%). The minimum inhibitory concentration and minimum fungicidal concentration of CMEO were determined as 421.7 ± 27.14 and 618.3 ± 79.35 ppm, respectively. The scanning electron microscopic observation of CMEO exposed macroconidia was exhibited a detrimental morphology with vesicles, craters, protuberance, and rough surfaces related to control fungi. The CMEO induced the death of fungi through elevating intracellular reactive oxygen species and lipid peroxidation, and depleting ergosterol content. Regrettably, essential oils are highly volatile and become unstable and lose their biological features on exposure to light, heat, pH, moisture, and oxygen. To overcome these issues, chitosan encapsulated CMEO nanoparticles (Ce-CMEO-NPs) were prepared. The synthesized Ce-CMEO-NPs have spherical morphology with Zeta potential of 39.3-37.2 mV and their corresponding size was found in range of 455-480 nm. The Fourier transform infrared analysis confirmed that bio-active constituents of CMEO were well stabilized due to chitosan conjugation and successfully formed Ce-CMEO-NPs. The in vitro release assay observed that the release of CMEO is stabilized due to the complex formation with chitosan and thereby, increases the lifetime antifungal activity of CMEO by gradual release of antifungal constituents of Ce-CMEO-NPs. In conclusion, antifungal and antimycotoxin activities of CMEO and Ce-CMEO-NPs against F. graminearum were assessed in maize grains under laboratory conditions over a storage period of 28 days. Interestingly, Ce-CMEO-NPs were presented efficient and enhanced antifungal and antimycotoxin activities related to CMEO, and it could be due to perseverance of antifungal activity by controlled release of antifungal constituents from Ce-CMEO-NPs. The study concluded that Ce-CMEO-NPs could be highly appropriate as mycobiocides in safeguarding the agricultural commodities during storage period in agricultural and food industries.

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Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

Publications

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

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