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Team:Toulouse/Project/Fungicides
From 2014.igem.org
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<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy minimizes the risk of the introduction of a resistance mechanism.</p> <br> | <p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy minimizes the risk of the introduction of a resistance mechanism.</p> <br> | ||
| - | <p class="textesimple"> | + | <p class="textesimple">Originally from plants, these peptides have different targets to maximize the lethality on <i>C. platani</i>.</p> |
<br></br> | <br></br> | ||
<ul> | <ul> | ||
| - | <li class="tree"><p class="texte"><b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi. </p></li> | + | <li class="tree"><p class="texte"><b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi.</p></li> |
| - | <li class="tree"><p class="texte"><b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and <i>in vitro</i> assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p></li> | + | <li class="tree"><p class="texte"><b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid <i>Gastrodia elata</i>, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and <i>in vitro</i> assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p></li> |
| - | <li class="tree"><p class="texte"><b>EcAMP-1 </b>(<i>Echinochloa crus-galli </i> AntiMicrobial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP-1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li> | + | <li class="tree"><p class="texte"><b>EcAMP-1 </b>(<i>Echinochloa crus-galli</i> AntiMicrobial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP-1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP-1 exhibits high activity against fungi of the genus <i>Fusarium</i>.</p></li> |
</ul> | </ul> | ||
</p> | </p> | ||
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<br> | <br> | ||
<p class="title2">Secretion</p> | <p class="title2">Secretion</p> | ||
| - | <p class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process. | + | <p class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.</p><br> |
Revision as of 15:38, 16 October 2014
Fungicides
To eradicate fungal diseases
Project > Fungicides
Figure 1: Scheme of the fungicide module
The main objective of SubtiTree is to ensure the destruction of the pathogenic fungi inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. This triple therapy minimizes the risk of the introduction of a resistance mechanism.
Originally from plants, these peptides have different targets to maximize the lethality on C. platani.
D4E1 is a synthetic peptide analog to Cecropin B AMPs (AntiMicrobial Peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi.
GAFP-1 (Gastrodia Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP-1 accumulates in nutritive corms where the fungal infection takes place, and in vitro assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.
EcAMP-1 (Echinochloa crus-galli AntiMicrobial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP-1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP-1 exhibits high activity against fungi of the genus Fusarium.
More information about this module
We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the three genes coding for the antifungal peptides are placed under the control of the constitutive promoter Pveg in Bacillus subtilis.
Figure 2: Fungicide operon
Added parts
EcAMP-1
EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (BBa_K1162001). This part has been modified and improved by our team (BBa_K1364019).
D4E1 and GAFP-1
We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (See Submitted parts).
We ordered the genes to a synthesis company and did cloning. These new BioBricks were designed in order to be expressed and secreted with Bacillus subtilis.
Secretion
In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process.
Figure 3: Design of GAFP-1 and D4E1
References
A. J De Lucca, J.M Bland, C. Grimm, T.J Jacks. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 . Canadian Journal of Microbiology. 1998, Vol. 44:514-520.
Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland. Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.
M. Visser, D. Stephan, J.M. Jaynes and J.T. Burger. A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.
K. D. Cox, D. R. Layne, R. Scorza, G Schnabel. Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco. Planta. 2006, Vol. 224:1373–1383
Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu and Willy Dillen. Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties. The Plant Journal. 2001, Vol. 25(6), 651±661
Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli). The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153
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