Hevein-Like Antimicrobial Peptides Wamps: Structure-Function Relationship in Antifungal Activity and Sensitization of Plant Pathogenic Fungi to Tebuconazole by WAMP-2-Derived Peptides.

Tatyana Odintsova,Vitaly Dzhavakhiya,Bakhyt Kartabaeva,Tatyana Pasechnik,Marina Slezina,Ekaterina Istomina,Larisa Shcherbakova

doi:10.3390/ijms21217912

Abstract

Hevein-like antimicrobial peptides (AMPs) comprise a family of plant AMPs with antifungal activity, which harbor a chitin-binding site involved in interactions with chitin of fungal cell walls. However, the mode of action of hevein-like AMPs remains poorly understood. This work reports the structure–function relationship in WAMPs—hevein-like AMPs found in wheat (Triticum kiharae Dorof. et Migush.) and later in other Poaceae species. The effect of WAMP homologues differing at position 34 and the antifungal activity of peptide fragments derived from the central, N- and C-terminal regions of one of the WAMPs, namely WAMP-2, on spore germination of different plant pathogenic fungi were studied. Additionally, the ability of WAMP-2-derived peptides to potentiate the fungicidal effect of tebuconazole, one of the triazole fungicides, towards five cereal-damaging fungi was explored in vitro by co-application of WAMP-2 fragments with Folicur® EC 250 (25% tebuconazole). The antifungal activity of WAMP homologues and WAMP-2-derived peptides varied depending on the fungus, suggesting multiple modes of action for WAMPs against diverse pathogens. Folicur® combined with the WAMP-2 fragments inhibited the spore germination at a much greater level than the fungicide alone, and the type of interactions was either synergistic or additive, depending on the target fungus and concentration combinations of the compounds. The combinations, which resulted in synergism and drastically enhanced the sensitivity to tebuconazole, were revealed for all five fungi by a checkerboard assay. The ability to synergistically interact with a fungicide and exacerbate the sensitivity of plant pathogenic fungi to a commercial antifungal agent is a novel and previously uninvestigated property of hevein-like AMPs.

Highlights

Despite the successful solution of many plant protection issues by using breeding for resistance and various green biotechnologies [1,2], fungal diseases continue to cause yield losses of economically important crops, including cereals [3], and treatment with chemical fungicides still remains the most relevant way to maintain a favorable phytosanitary situation in the field and to guarantee high yields.At the same time, it is well known that the use of fungicides is associated with ecological risks, and their toxic residues in forage and food products can cause problems for animal husbandry and human health
We modeled the tertiary structures of WAMP homologues using as a template WAMP-1a, whose solution structure was determined by NMR spectroscopy [16] (Figure 2)
Wheat-pathogenic Fusarium culmorum OR-02-37, F. avenaceum Br-04-60, Alternaria alternata MRD1-12, and barley-damaging Bipolaris sorokiniana KrD-81 were obtained from the State Collection of Plant Pathogenic Microorganisms at the All-Russian Research Institute of Phytopathology (ARRIP), while cucumber-damaging Cladosporium cucumerinum C5, and wheat kernels artificially infested with Parastagonospora nodorum B-9/47 were kindly supplied by the ARRIP Departments of Molecular Biology, and Mycology, respectively, and F. oxysporum F37 pathogenic for tomato was from the working collection of the ARRIP Laboratory of Physiological Plant Pathology

Summary

Introduction

Despite the successful solution of many plant protection issues by using breeding for resistance and various green biotechnologies [1,2], fungal diseases continue to cause yield losses of economically important crops, including cereals [3], and treatment with chemical fungicides still remains the most relevant way to maintain a favorable phytosanitary situation in the field and to guarantee high yields.At the same time, it is well known that the use of fungicides is associated with ecological risks, and their toxic residues in forage and food products can cause problems for animal husbandry and human health. Et Migush., we isolated two unique hevein-like AMPs named WAMP-1a and 1b, which differ by a single C-terminal arginine residue [15] They contain 44 and 45 amino acid residues, respectively, and are positively charged [15]. Analyzing the biological diversity of wamp genes in Poaceae plants, we discovered high conservation of the amino acid sequences of the mature WAMPs with a single variable position 34, in which several amino acids were found: Ala, Lys, Asn, Glu and Val [17,19] This residue is located in loop 6 (loops are regions between the adjacent cysteine residues) between the alpha-helix (residues 29–32) and beta-strand 4 (residues 36–39) (Figure 1) [16]. Our discovery that WAMPs, which do not inhibit fungalysin, still effectively inhibit different fungi in vitro suggests the existence of an alternative mode of action

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