Team:Toulouse/Project/Fungicides

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.

Originated from plants, these peptides have different target to maximize the lethality on C. platani.

- D4E1 is a synthetic peptide anlogo 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. GAFP1 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 Anti Microbial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP1 exhibits high activity against fungi of the genus Fusarium.

More information on this module

Final constructions: (see Parts to have more details on the intermediate parts) We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the 3 genes are placed under the control of a constitutive promoter in Bacillus subtilis: Pveg.

EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (BB_K1162001). We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (see parts). 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.

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

Binding

Spreading