Despite the absence of disulfide bonds in its linear

Despite the absence of disulfide bonds in its linear structure, KT43C displays antifungal and antibacterial activity. Disulfide bonds are determinants of defensins' integrity and have been reported to increase the antimicrobial activity (Jenssen et al., 2006). However, some linear derivatives of AMPs (Wu and Hancock, 1999) and defensins (Liu et al., 2008) are able to retain partial or complete antimicrobial activity after removal of the disulfide bonds. Other parameters, such as net charge, hydrophobicity, amphipathicity and flexibility, are essential for the antimicrobial activity of AMPs. Although disulfide bridges are not essential for the antimicrobial and antifungal activity of Cp-thionin II, the presence of free cysteine residues may modify the hydrophobicity of the peptide and then change its activity. However, this effect seems unclear for linear analogues of defensins (Liu et al., 2008, Nagano et al., 1999). Dimerization of plant defensins is also a highly significant criterion for their antifungal activity (Song et al., 2011). Plant defensins that form dimers become highly efficient molecules against pathogenic fungi due to a stronger interaction with the negatively charged proteins of the fungal Cholera Toxin and membrane (Lay and Anderson, 2005). However, the oligomerization of defensins does not appear to be crucial, as shown for the antibacterial activity of Cp-thionin II (Franco et al., 2006). KT43C displayed antifungal activity against F. culmorum, A. niger and P. expansum. These three fungal species belong to the same subdivision, Pezizomycotina, but F. culmorum belongs the class of Sordariomycetes while P. expansum and A. niger both belong to the class of Eurotiomycetes. Although the mechanistic action of KT43C on these fungi may have similarities, differences in cell/wall composition or fungal physiology between these species may be pertinent to the mode of action of KT43C and its antifungal potency. Differences in the mode of action of the plant defensin MtDef4 against Neurospora crassa and F. graminearum have been described by El-Mounadi et al. (2016). KT43C inhibited growth of F. culmorum without inducing morphogenic changes in the hyphae (Fig. 3). This finding is in agreement with the ability of KT43C to inhibit Gram-positive bacteria (Kraszewska et al., 2016) and the antibacterial activity of native peptide (Franco et al., 2006). Indeed, only non-morphogenic defensins appear to have an effect on bacteria (Carvalho and Gomes, 2009). After heat treatment, KT43C retained its antifungal activity against F. culmorum. The heat stability of KT43C has also previously been shown regarding its antibacterial potency (Kraszewska et al., 2016). Terras et al. (1992) and Broekaert et al. (1995) have reported the thermal stability of defensins from radish and other plant species. The role of disulfide bonds in defensins stabilization was highlighted by Terras et al. (1992). KT43C appeared unstructured in aqueous solutions but adopts an α-helical conformation in a membrane environment (Fig. 7). The random conformations and flexibility of the peptide could protect from thermal denaturation. The adoption of a structured conformation in presence of SDS may indicate possible interactions between KT43C and a membrane-mimic environment. This new conformation could be related to its antifungal activity (Domingues et al., 2008, Liu et al., 2008). The presence of ions, especially divalent cations, has been proven to decrease the antifungal activity of native plant defensins (Vriens et al., 2014). The antifungal activity of this linear analogue of Cp-thionin II was demonstrated to be also affected by the presence of cations (Fig. 4). Kraszewska et al. (2016) reported that the peptide keeps its antibacterial activity in the presence of NaCl, up to 50 mM, but loses it at 100 mM. The loss of activity in presence of cations is a common feature for plant defensins and AMPs linear derivatives in general (Adem Bahar and Ren, 2013, Vriens et al., 2014). This effect is due to the weakening of electrostatic interactions between the cationic peptides and the negatively charged membrane of microbial cells (Wu et al., 2003). Other potential effects may include structural changes in the peptide (Oard and Karki, 2006), or stabilization of the microbial membrane by cations (Thevissen et al., 1999).