研究领域：生物化学 植物病理学

关键字：抗真菌活性 抗真菌多肽 非常规多肽 未注释的编码序列 植物病原真菌 广谱的

多肽通常由 2-100 个氨基酸残基组成，是许多生理过程的关键调节因子（Tavormina 等，2015；Wang 等，2020a）。常规多肽已被发现具有显著的抗真菌活性，且对环境和人类健康相对安全（Marcos 等，2008；Ribeiro 等，2013）。因此，抗真菌多肽作为新型杀菌剂，是应对植物病原微生物抗生素耐药性日益增加的有前景的替代品。最近，一类源自先前未注释的编码序列（CDSs）的新型多肽——非常规多肽（NCPs），引起了广泛关注（Wang 等，2020b）。研究表明，NCPs 在各种生物过程中发挥着重要作用（Khitun 等，2019；Plaza 等，2017）。然而，迄今为止，尚未有关于 NCPs 抗真菌活性的报道。Peptides are generally composed of 2-100 amino acid residues and well known as key regulators of many physiological processes (Tavormina et al., 2015; Wang et al., 2020a). Conventional peptides have been found to exhibit pronounced antifungal activities and are relatively safe for the environment and human health (Marcos et al., 2008; Ribeiro et al., 2013). Therefore, antifungal peptides, as novel fungicides, are promising alterna tives for combating the increased incidence of antibiotic resis tance in plant pathogenic microbes. Recently, non-conventional peptides (NCPs), a novel class of peptides derived from previously unannotated CDSs, have attracted significant attention (Wang et al., 2020b). Studies have demonstrated that NCPs play essential roles in various biological processes (Khitun et al., 2019; Plaza et al., 2017). However, antifungal activity of NCPs has not been reported to date.

植物病原真菌是一类重要的微生物群，持续给全球农业生产造成巨大的经济损失（Moller 和 Stukenbrock，2017）。为了探索 NCPs 对植物病原真菌的抗真菌作用，我们相应地合成了 246 种 NCPs（Bioyears，中国武汉）。这些 NCPs 是在三叶期的正常生长条件下，通过整合的肽基因组学流程从玉米自交系 B73 的叶片中鉴定出来的（Wang 等，2020b）。它们源自基因间区、非翻译区（UTRs）、内含子、连接区以及移码外显子（图 1a）。本研究测试了玉蜀黍平脐蠕孢（Bipolaris zeicola）、弯孢霉（Curvularia lunata）、禾谷镰刀菌（Fusarium graminearum）和玉米平脐蠕孢（Bipolaris maydis）这几种病原菌。这些病原菌会导致全球栽培作物发生严重的真菌病害。如图 1a 所示，通过菌丝生长速率法评估了这些 NCPs 对四种病原真菌的抗真菌活性（Agarwal 等，2001）。简而言之，将 NCPs 溶液加入马铃薯葡萄糖琼脂（PDA）培养基中。从真菌菌落边缘取 5 mm 的真菌接种块置于 PDA 培养基中央，并在 28 °C 黑暗条件下培养。在接种后 12、24 和 36 小时（hpi）记录菌丝菌落直径和菌丝形态。每个处理重复三次。菌丝生长抑制率通过测量菌落直径确定，并按以下公式计算：I% = /(C-d) × 100%。Fungal plant pathogens comprise an important group of microorganisms that continuously cause substantial economic losses in agricultural production around the world (Moller and Stukenbrock, 2017). To explore the antifungal effect of NCPs against plant pathogenic fungi, 246 NCPs were synthesized accordingly (Bioyears, Wuhan, China). These NCPs were identified from the leaves of maize inbred line B73 by an integrated peptidogenomic pipeline under normal growth condition at the three-leaf stage (Wang et al., 2020b). They are derived from intergenic regions, untranslated regions (UTRs), introns, junctions and out-of-frame exons (Figure 1a). The pathogens Bipolaris zeicola, Curvularia lunata, Fusarium graminearum and Bipolaris maydis were tested in this study. These pathogens cause major fungal diseases in cultivated crops worldwide. The antifungal activity of these NCPs against four pathogenic fungi was evaluated by the mycelium growth rate method (Agarwal et al., 2001), as shown in Figure 1a. In brief, NCP solutions were added to the potato dextrose agar (PDA) medium. A 5 mm plug of fungal inoculum from the edge of fungal colony was placed at the centre of the PDA medium and incubated at 28 °C in the dark. The diameter of the mycelial colony and hyphal morphology were recorded at 12, 24 and 36 hours post inoculation (hpi). Each treatment was performed in triplicate. The mycelium growth inhibition rate was determined by measuring the colony diame ters and calculated by the following equation: I% = [(C-d)- (T-d)]/ (C-d) 9 100%.