Fernández Agustina, Colombo María Laura, Curto Lucrecia M, Gómez Gabriela E, Delfino José M, Guzmán Fanny, Bakás Laura, Malbrán Ismael, Vairo-Cavalli Sandra E
CIPROVE-Centro Asociado CIC, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Front Microbiol. 2021 Feb 17;12:632008. doi: 10.3389/fmicb.2021.632008. eCollection 2021.
is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant . The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAP and SmAP) and two from the γ-core region (SmAP and SmAP). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAP, SmAP, and SmAP inhibited the growth of the phytopathogen at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.
是小麦赤霉病（FHB）的病原体，这种疾病会导致小麦作物产量大幅下降，而受感染谷物中存在的霉菌毒素会进一步加剧这种情况，这些霉菌毒素可能会给人类和动物带来健康问题。植物防御素和类防御素蛋白是抗菌肽（AMPs）；它们是小的碱性、富含半胱氨酸的肽（CRPs），在植物界普遍表达，主要参与宿主防御。它们具有高度可变的序列但结构保守。位于植物防御素C端区域的γ-核心具有保守的β-发夹结构，是含二硫键的抗菌肽中抗菌活性的一个众所周知的决定因素。植物防御素的另一个保守基序是位于N端区域的α-核心，在含二硫键的抗菌肽中不保守，尚未得到广泛研究。在本报告中，我们克隆了在野生植物花中表达的假定抗菌蛋白DefSm2。该cDNA编码一种具有两个融合碱性结构域的蛋白质，一个N端防御素结构域（DefSm2-D）和一个C端富含精氨酸和赖氨酸的结构域。为了进一步表征DefSm2-D结构域，我们构建了一个基于3D模板的模型，该模型将有助于支持新型抗真菌肽的设计。我们设计了四种潜在的抗真菌肽：两种来自DefSm2-Dα-核心区域（SmAP和SmAP），两种来自γ-核心区域（SmAP和SmAP）。我们化学合成并纯化了这些肽，并通过电喷雾电离质谱（ESI-MS）和圆二色性（CD）光谱对它们进行了进一步表征。SmAP、SmAP和SmAP在低微摩尔浓度下抑制了植物病原体的生长。分生孢子暴露于肽的杀菌浓度会导致荧光探针碘化丙啶（PI）的膜通透性增加，这表明这是真菌细胞死亡的主要促成因素之一。此外，通过透射电子显微镜（TEM）观察，处理0.5小时的分生孢子显示出细胞质紊乱。值得注意的是，源自α-核心的肽在分生孢子细胞壁上诱导了形态变化，这是一个有前景的靶点，因为其独特的生化和结构组织在植物和哺乳动物细胞中不存在。
