EcAMP3

Herein, we describe a modified form of the antimicrobial hairpin-like peptide EcAMP1, isolated from barnyard grass (E. crusgalli) seeds, which is structurally characterized by a combination of high-pressure liquid chromatography, mass spectrometry, and automated Edman sequencing. This derivate has a single amino acid substitution (Pro19Hyp) in the second α-helical region of the molecule, which is critical for the formation of the hydrophobic core and the secondary structure elements. Comparing the antifungal activity of these two peptides, we found that the modified EcAMP1-Hyp had a significantly weaker activity towards the most-sensitive plant pathogenic fungus Fusarium solani. Molecular dynamics simulations and in vitro binding to the commercial polysaccharides allowed us to conclude that the Pro-19 residue is important for binding to carbohydrates located in the spore cell wall and it chiefly exhibits a fungistatic action representing the hyphal growth inhibition. These data are novel and significant for understanding a role of α-hairpinins in plant immunity.

An interaction of recombinant hairpin-like cationic peptide EcAMP1 with conidia of plant pathogenic fungus Fusarium solani at the cellular level was studied by a combination of microscopic methods. EcAMP1 is from barnyard grass (Echinochloa crusgalli L.), and obtained by heterologous expression in Escherichia coli system. As a result, a direct relationship between hyphal growth inhibition and increasing active peptide concentration, time of incubation and fungal physiological condition has been determined. Dynamics of accumulation and redistribution of the peptide studied on fungal cellular cover and inside the conidia cells has been shown. The dynamics are dependent on time of coupling, as well as, a dissimilarity of EcAMP1 binding with cover of fungal conidia and its stepwise accumulation and diffuse localization in the cytoplasm. Correlation between structural disruption of fungal conidia and the presence of morphological changes has also been found. The correlation was found under the influence of peptide high concentrations at concentrations above 32 μM. The results indicate the presence of a binding of EcAMP1 with the surface of fungal conidia, thus, demonstrating a main specificity for its antifungal action at the cellular level. These results, however, cannot exclude the existence of attendant EcAMP1 action based on its intracellular localization on some specific targets. SCANNING 38:591-598, 2016. © 2016 Wiley Periodicals, Inc.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.