Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αβ structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2–S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

防御素是在先天免疫系统中起作用的抗菌肽。OsAFP1是一种从稻（稻）中鉴定出的防御素，对水稻病原体具有抗菌活性。有趣的是，OsAFP1还显示出对人类病原真菌白色念珠菌的有效抗真菌活性。通过诱导靶细胞凋亡，表明OsAFP1代表了潜在的新型抗生素候选物。然而，需要进一步的分析，尤其是在结构水平上的分析，以阐明OsAFP1抗真菌活性的机理基础。在这里，我们使用X射线晶体学确定了OsAFP1的三维结构。OsAFP1具有在植物防​​御素中高度保守的半胱氨酸稳定的αβ结构，并呈现出似乎是抗真菌活性所必需的二聚体结构。OsAFP1结构与烟草的重叠NaD1与磷脂酸复合表明目标分子很可能被困在每个OsAFP1二聚体的S2-S3环之间。在使用固定有各种膜脂质成分的硝酸纤维素膜进行的脂质结合分析中，发现OsAFP1与带有磷酸基团的磷脂酰肌醇（PIP）结合，尤其是PI（3）P。这些结果表明，OsAFP1通过与白色念珠菌细胞膜中包含的PI（3）P结合而发挥抗真菌活性，从而施加细胞应激并诱导细胞凋亡。此外，OsAFP1的结构和位点特异性突变分析显示，Arg1，His2，Leu4，Arg9和Phe10在OsAFP1二聚体形成中起关键作用。因此，我们的研究为OsAFP1的抗真菌机制提供了新颖的见解。