Identification and functional characterization of a glycosylceramide Delta 3-(E) desaturase from phytopathogenic fungi

鞘糖脂是真核生物中普遍存在的膜脂。它们的神经酰胺主干可以在许多方面进行修饰。在哺乳动物和酵母细胞中，这些脂类参与细胞周期进程、细胞凋亡、病原体防御和应激反应，而在真菌中，只有葡萄糖神经酰胺作为激发子和植物防御素的靶标，在这些生物中也具有类似的功能。一些真菌糖基神经酰胺含有一种3-(E)-不饱和脂肪酸，其生物学功能尚不清楚。到目前为止，还没有从任何生物体中鉴定出一种去饱和酶，它在脂肪酸残基的3位引入了一个双键。在我们之前从植物和真菌中克隆脂肪酸和狮身人面像脱氢酶的研究过程中，我们现在已经用生物信息学的方法在植物病原真菌Fusum gramineum和Magnaporthe grisea的基因组中找到了合适的候选序列。这些序列很可能编码糖基神经酰胺？3-(E)-去饱和酶。在PCR扩增后，这些基因的功能鉴定应通过在毕赤酵母中的异源表达来进行。此外，这两种谷物病原真菌都适合通过同源重组敲除基因，并作为植物防御素的靶标。这将使我们能够研究这种3-去饱和酶在这些植物病原真菌中的功能及其对它们对小麦、大麦、玉米和水稻等谷物的毒力的贡献。

Glycosphingolipids are ubiquitous membrane lipids present in all eukaryotic organisms. Their ceramide backbone may be modified in many respects. In mammalian und yeast cells, these lipids are involved in cell cycle progression, apoptosis, pathogen defense and stress response, while in fungi only the effects of glucosylceramides as elicitors and as a target for plant defensins point to similar functions also in these organisms. Some fungal glycosylceramides contain a ¿3-(E)-desaturated fatty acid, the biological function of which is still unknown. A desaturase, which introduces a double bond into the ¿3-position of a fatty acid residue, has not been identified from any organism so far. In the course of our previous research on the cloning of fatty acid and sphingobase desaturases from plants and fungi, we have now identified suitable candidate sequences in the genomes of the phytopathogenic fungi Fusarium gramineum und Magnaporthe grisea in a bioinformatic approach. These sequences most likely code for a glycosylceramide ¿3-(E)-desaturase. Following PCR amplification, the functional identification of these genes should be carried out by heterologous expression in the yeast Pichia pastoris. Moreover, both cerealpathogenic fungi are suitable for a gene knock-out by homologous recombination and as a target for plant defensins. This will enable us to study the function of this ¿3-desaturase in these phytopathogenic fungi and its contribution to their virulence towards cereals such as wheat, barley, maize and rice.