Aerts An M, François Isabelle E J A, Meert Els M K, Li Qiu-Tian, Cammue Bruno P A, Thevissen Karin
Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Heverlee, Belgium.
J Mol Microbiol Biotechnol. 2007;13(4):243-7. doi: 10.1159/000104753.
RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant DeltagcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.
RsAFP2（萝卜抗真菌肽2）是一种从萝卜种子中分离出来的抗真菌植物防御素，它与敏感酵母和真菌细胞膜中的葡糖神经酰胺（GlcCer）相互作用，诱导膜通透性增加和真菌细胞死亡。然而，使用含有纯化GlcCer的羧基荧光素标记的小单层囊泡，我们并未观察到RsAFP2插入此类囊泡后导致的通透性增加。因此，我们将重点放在了RsAFP2与真菌细胞膜中GlcCer结合后下游可能的信号级联反应上。我们发现RsAFP2以剂量依赖的方式在白色念珠菌野生型中诱导活性氧（ROS）产生，但在其膜中缺乏RsAFP2结合位点的RsAFP2抗性白色念珠菌ΔgcsC突变体中则完全不诱导。这些发现表明RsAFP2与GlcCer的上游结合是导致酵母细胞死亡的ROS产生所必需的。此外，抗氧化剂抗坏血酸可阻断RsAFP2诱导的ROS生成以及RsAFP2的抗真菌活性。这些数据表明存在一种细胞内植物防御素诱导的信号级联反应，该反应涉及ROS生成并导致真菌细胞生长停滞。
