Molecular analysis of arthrosporol, ascaroside and nutritional signaling during the interaction of the nematode-trapping fungus Duddingtonia flagrans with Caenorhabditis elegans

有机体的相互作用对地球上的生命至关重要。这种相互作用通常是非常复杂的，并在进化过程中形成。在真菌相互作用的情况下，它们可以从共生到致病再到捕食。我们正在研究捕食线虫的真菌，它们会产生不同的诱捕装置，将线虫引诱到陷阱中，用穿透菌丝入侵线虫，然后在整个蠕虫体内定居。我们的模型是Duddintia flagrans，它在实验室生长得很好，可以产生粘性诱捕网。鞭毛虫在畜牧业中也被用作生防剂，并有可能在未来进一步开发用于植物病原线虫的防治。与其他捕食线虫的真菌不同的是，它能产生抗性厚壁孢子，可用于土壤应用。真菌-线虫相互作用的第一步之一是真菌对线虫的感知和陷阱形成的启动。由于陷阱的形成只有在存在一定数量的线虫而其他营养物质不可用的情况下才有意义，因此一种基于低分子化合物的有趣的有机体间通讯系统应运而生。次生代谢物、不同节孢素和6-甲基水杨酸可抑制陷阱的形成。这些真菌能够感觉到蛔虫苷--重要的线虫分子--从而感觉到线虫的存在。蛔虫苷抑制真菌衍生的负性信号分子的产生，从而导致陷阱的诱导。该系统类似于群体感知机制，使线虫密度的感知成为可能。在当前的研究方案中，将分析陷阱诱导和形态发生背后的信号级联。初步数据表明，G蛋白和G蛋白偶联膜受体(GPCRs)参与了营养状态和线虫存在的整合。这些信号级联将通过基因缺失和过度表达实验来解决。我们将验证这一假设是否通过水平基因转移(HGT)从线虫中获得了一些真菌蛔虫苷类GPCR。除了通过基因缺失、基因过度表达和使用结构性G-α等位基因来阐明信号级联的组成外，G-蛋白与其GPCR的相互作用将通过双分子荧光互补(BIFC)、酵母双杂交和FRET分析在时间和空间上进行研究。为了进一步阐明TRAP诱导和TRAP形态发生的过程，将分离信号级联的靶基因，并在全基因组转录分析后对其进行鉴定。

Organismic interactions are crucial for live on earth. Such interactions are often very sophisticated and have been shaped during evolution. In the case of fungal interactions, they can range from symbiotic to pathogenic to predatory. We are studying nematode-trapping, predatory fungi, which produce different trapping devices, lure nematodes into the traps, invade the nematodes with penetrating hyphae and subsequently colonize the entire worm body. Our model is Duddintonia flagrans, which grows well in the lab and produces adhesive trap networks. D. flagrans is also used as a biocontrol agent in livestock farming, and may be further developed against plant pathogenic nematodes in the future. As a difference to other nematode-trapping fungi, it produces resistant chlamydospores, which may be used for its application in soil. One of the first steps of the fungal-nematode interaction is the fungal sensing of the nematodes and the initiation of trap formation. Because trap formation only makes sense, if a certain number of nematodes is present and other nutrients are not available, an interesting interorganismic communication system based on low-molecular weight compounds evolved. Trap formation is inhibited by secondary metabolites, different arthrosporols and 6-methyl salicylic acid. The fungi are able to sense ascarosides – important nematode molecules – and thereby the presence of nematodes. Ascarosides inhibit the production of the fungal-derived, negative signaling molecules, which leads to trap induction. The system resembles a quorum sensing mechanism, enabling the sensing of the nematode density.In the current research proposal, the signaling cascades underlying trap induction and morphogenesis will be analyzed. Preliminary data suggest the involvement of G-proteins and G-protein coupled membrane receptors (GPCRs) in the integration of the nutritional status and the presence of nematodes. The signaling cascades will be resolved by gene deletion and overexpression experiments. We will test the hypothesis if some fungal ascaroside-sensing GPCRs were acquired by horizontal gene transfer (HGT) from nematodes during evolution.Besides the elucidation of the components of the signaling cascades by gene deletion, gene overexpression and the use of constitutive-active G-alpha alleles, the interaction of the G-proteins with their GPCRs will be studied in time and space by bimolecular fluorescence complementation (BiFC), yeast-two-hybrid and FRET analyses.To further elucidate the process of trap induction and trap morphogenesis, target genes of the signaling cascades will be isolated and characterized after genome-wide transcription analyses.