Team:Toulouse/Project/Fungicides

Figure 1: Schema 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 provides the advantage to minimize the resistance phenomenon.

Originally from plants, these peptides have different targets thus increasing 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. The three genes coding for the antifungal peptides are placed under the control of the constitutive promoter P_veg 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) with the addition of a STOP codon after the coding sequence.

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 sequences of D4E1 and GAFP-1 were 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

Binding

Spreading