Studying of cellular interaction of hairpin‐like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds with plant pathogenic fungus Fusarium solani using microscopy techniques

Abstract

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.

Characterization of Hydroxyproline-Containing Hairpin-Like Antimicrobial Peptide EcAMP1-Hyp from Barnyard Grass (Echinochloa crusgalli L.) Seeds: Structural Identification and Comparative Analysis of Antifungal Activity

“…After lyophilization, 4.2 mg of the peptide, with oxidized SH-groups, was obtained. Recombinant EcAMP1 was received in heterologous expression in Escherichia coli , as mentioned earlier [ 22 ].…”

“…This modified amino acid residue was determined by the N-terminal Edman sequencing, followed by a comparison with a non-coded l -hydroxyproline analytical standard. The detected peptide possessed weaker antifungal activity towards the plant pathogenic fungus F. solani , one of the most EcAMP1-sensitive cultures [ 12 , 22 ]. It is interesting that only a single amino acid substitution (Pro19Hyp) was able to reduce the antifungal effect.…”

“…It is typical that barnyard grass, which is represented as a annual cereal, is a donor for the whole family of α-hairpinins, titled EcAMPs. These peptides were involved in two highly active fungistatic molecules, EcAMP1 [ 12 , 22 ] and EcAMP3 [ 23 ], and EcAMP2, which is not active, at all [ 24 ]. None of the peptides was found to have any posttranslational modifications, excepting disulfides.…”

“…In the short term (1–6 h), most of the peptide quantity accumulated onto the spore cover (the cell wall and plasma membrane, which could not be differentiated using low-resolution optical microscopy techniques, 130 nm maximum). Subsequently, some of the peptide was internalized into the cytosol [ 12 , 22 ]. We suggest that, at least, one mode of the peptide action was associated with fungal spore cover, mainly cell wall consisting of carbohydrates (chitin, β-1,3/1,6 glucans, generally), and therefore, we conducted in vitro binding of both peptides to commercially available chitin and β-1,3 glucans.…”

Primary Structure Analysis of Antifungal Peptides from Cultivated and Wild Cereals

“…For the first time in wild cereals since the description of MBP-1, a novel family of antimicrobial hairpin-like peptides (EcAMPs) was discovered in barnyard grass ( E. crusgalli ) [ 94 , 95 , 96 ], as well as in the sequel that was also in cultivated wheat ( T. kiharae ) [ 97 ]. Studying the biological activity mechanisms of these peptides at the cellular level allowed us to conclude about their fungistatic influence on microscopic fungi, which was implemented as a delay in the spore germination and growth power of hyphae [ 94 , 98 ]. The data on the quantitative antifungal activity of some hairpin-like peptides are presented in Table 3 .…”