A Protein from the Mold <i>Aspergillus giganteus</i> Is a Potent Inhibitor of Fungal Plant Pathogens

Vila, Laura; Lacadena, Valle; Fontanet, P.; Martínez‐del‐Pozo, Álvaro; Segundo, Blanca San

doi:10.1094/mpmi.2001.14.11.1327

Cited by 55 publications

(32 citation statements)

References 12 publications

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“…Since AFP is an extracellular protein, the potential involvement of its two binding abilities, to membranes and DNA, in its antifungal action should be considered. In regard to this, it has been reported that AFP completely inhibits the growth of phytopathogenic fungi in rice with no toxicity toward plant protoplasts (38) and that heterologous expression of the afp gene enhanced fungal resistance in transgenic wheat (39), which opens the potential use of the afp gene to enhance crop protection against fungal pathogens in transgenic plants. Finally, the activity of AFP on promoting DNA condensation and interacting with lipid membranes may also be useful in designing non-viral gene delivery systems.…”

Section: Resultsmentioning

confidence: 99%

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Pozo

Lacadena²,

Mancheño

et al. 2002

Journal of Biological Chemistry

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The antifungal protein AFP is a small polypeptide of 51 amino acid residues secreted by Aspergillus giganteus. Its potent activity against phytopathogenic fungi converts AFP in a promising tool in plant protection. However, no data have been reported regarding the mode of action of AFP. The three-dimensional structure of this protein, a small and compact ␤ barrel composed of five highly twisted antiparallel ␤ strands, displays the characteristic features of the oligonucleotide/oligosaccharide binding (OB fold) structural motif. A comparison of the structures of AFP and OB fold-containing proteins shows this structural similarity despite the absence of any significant sequence similarity. AFP, like most OB fold-containing proteins, binds nucleic acids. The protein promotes charge neutralization and condensation of DNA as demonstrated by electrophoretic mobility shift and ethidium bromide displacement assays. Nucleic acid produces quenching of the protein fluorescence emission. This nucleic acid interacting ability of AFP may be related to the antifungal activity of this small polypeptide.The mold Aspergillus giganteus produces two major extracellular proteins, ␣-sarcin, a potent cytotoxic ribonuclease, and the so-called antifungal protein (AFP) 1 (1-3). AFP is a small polypeptide of 51 amino acid residues that is secreted under the form of a rather inactive larger precursor containing 6 extra amino acid residues at the NH 2 -terminal end, which is processed in the extracellular medium (4). The amino acid and cDNA sequences of AFP have been reported, revealing a high content of disulfide bridges (fours bonds) and tyrosines and lysines (6 and 12 residues, respectively) (5-7). AFP shows a significant degree of sequence similarity only with both the abundantly secreted antifungal PAF protein (55 amino acid residues) from Penicillium chrysogenum (47% sequence identity) (8) and the antifungal peptide Anafp (56 amino acid residues) secreted by Aspergillus niger (31% sequence identity) (9). AFP has been tested, at concentrations as large as 0.2 mM, against a wide variety of microorganisms, including prokaryotes and eukaryotes (3). This protein inhibits the growth of some filamentous fungi, the minimal protein concentration for total inhibition being in the range of 6 -25 M (3), but it does not promote any effect on the producing mold or on bacteria or yeast. AFP is not active against P. chrysogenum or A. niger (3), the organisms producing PAF and Anapf proteins, respectively. Although these three proteins have been described as antifungal molecules, no data regarding their mode of action have been so far reported. We herein report the interaction of AFP with DNA. EXPERIMENTAL PROCEDURESUnless otherwise stated, all materials and reagents were molecular biology grade. Calf thymus double-stranded DNA (dsDNA) was purified as described previously (10, 11). The DNA fraction selected for the experiments described in this work (kept at Ϫ20°C until used) was that containing 200 bp dsDNA on average according to their electrophoret...

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Section: Resultsmentioning

confidence: 99%

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Pozo

Lacadena²,

Mancheño

et al. 2002

Journal of Biological Chemistry

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“…The inhibition zones which mediated by AFP as different concentrations demonstrated a positive relation between the concentration and the activity (Figure 5). This may be due to the influence on AFP hyphal extension by localisation on the outer membrane within defined areas cause short, thick and highly septated hyphae with constricted apical regions extruding from condensed mycelia [12] [29]. However, AFP shares several structural features with membrane acting proteins, such as an amphiphitic structure, a basic PI and the occurrence of eight cysteine residues, all involved in intermolecular disulfide bridges.…”

Section: Discussionmentioning

confidence: 99%

“…One thousand spore or conidia were added to 200 µl of culture medium containing AFP at different concentrations ranged from 0 -50 µg•ml −1 in microtiter plate (Greiner, Frickenhausen). After 72 h of incubation with continuous agitation at 120 rpm, the mycelium from each concentration was washed with phosphate buffer (pH, 7.0) then fixed with Poly-L-Lysine (PLL) on glass slid and covered with slid cover [29]. The morphological attributes were microscopically diagnosed.…”

Section: Determination Of the Morphological Changesmentioning

confidence: 99%

Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

Barakat¹

2014

FNS

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Interestingly, antifungal protein AFP was purified from Aspergillus giganteus supernatants with modified isoelectric focusing procedure after adaptation of the secretion conditions. Subsequently, the antifungal activity as well as the mode of action against Alternaria alternata was tested in vitro. Moreover, different concentrations of AFP were applied to banana fruits for 15 days at 20˚C in vivo. Obtained results illustrated that A. giganteus was able to secrete about 39.78 ± 2.39 mg AFP•l −1 Olson medium. The employed ammonium sulfate (AS 75%) precipitation procedure followed by dialysis steps yielded about 16 -22 mg AFP•l −1 culture supernatant with general mean of 18.67 ± 1.98 mg•l −1 . The lost amount of AFP during purification using AS and 3KDa cut-off dialysis membrane is about 50% thus, purification procedure must be further improved. Indeed, concluded results from MIC and hyphal extension inhibition test noticed that AFP was efficiently affected by either growth or hyphae form of A. alternata in vitro. The MIC of AFP against A. alternata was 2 μg•ml −1 . However, short, thick and highly septated hyphae with damaged constricted apical regions extruding from condensed mycelium aggregates in treated hyphae compared to the untreated culture was remarkably shown. The mode of action of in vitro experiment manifested that AFP was effective to act the fungal cell and permeabilize the cell membrane of A. alternata. Furthermore, the in vivo experiment showed that AFP could reduce post-harvest decay on banana caused by A. alternata. AFP at concentration of 15 and 25 μg•ml −1 exhibit Alternaria decayed reduction by 45.45% and 77.27%, respectively. While no Alternaria decayed area was observed when 50 μg•ml −1 was applied during the storage time. Quantification of DNA by species-specific PCR could exude a positive correlation between the DNA amount and decayed area. In conclusion, AFP can be efficiently used as a bio-preservative agent during storage and handling of banana fruits, and considered as an excellent biological alternative to combat secondary growth of filamentous fungi.

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“…The in vitro antifungal activity of both P. pastoris-produced recombinant AFPs, as well as the A. giganteus-produced natural AFP, was determined using a microtiter plate assay as previously described [13,15,28]. Briefly, 90 μl of a fungal spore suspension at 10 5 spores/ml in PDB (potato dextrose broth; DIFCO, Detroit, MI, USA), containing 0.003% (wt/vol) chloramphenicol, was allowed to pre-germinate for 6 h at 28°C.…”

Section: In Vitro Antifungal Activity Of the Purified Proteinsmentioning

confidence: 99%

“…One of these promising polypeptides is the antifungal protein (AFP) secreted by the mould Aspergillus giganteus. AFP is a basic, low molecular weight (51 residues long) protein showing in vitro antifungal properties against important plant pathogens, including Magnaporthe oryzae, Fusarium verticilloides, Phytophthora infestans, and Botrytis cinerea [11][12][13][14][15]. These fungi are responsible for important diseases in a large number of plants with agricultural and economical importance, such as rice, wheat, potato, or tomato.…”

Section: Introductionmentioning

confidence: 99%

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

López-Garcı́a

Moreno

Segundo

et al. 2010

Protein Expression and Purification

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The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (If-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP*) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. AFP* produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will allow getting further insights into the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches

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A Protein from the Mold Aspergillus giganteus Is a Potent Inhibitor of Fungal Plant Pathogens

Cited by 55 publications

References 12 publications

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

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A Protein from the Mold Aspergillus giganteus Is a Potent Inhibitor of Fungal Plant Pathogens

Cited by 55 publications

References 12 publications

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

The Antifungal Protein AFP of Aspergillus giganteusIs an Oligonucleotide/Oligosaccharide Binding (OB) Fold-containing Protein That Produces Condensation of DNA

Bio-Control of &lt;i&gt;Alternaria alternata&lt;/i&gt; during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

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Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique