Molecular Analysis of Necrotrophic Mycoparasitism in the Predator Yeast Saccharomycopsis schoenii

我们的工作旨在对舍氏酵母菌的捕食行为进行分子分析。酵母菌种类具有强大的潜力，因为：(1)它们易于培养，是单倍体（我们未发表的结果），是同thalic或异thalic，并且可以进行正向和反向遗传。（ii）它们在酵母菌中显示出坏死性分枝寄生的独特特性，为研究宿主-病原体相互作用提供了一个简单的模型，例如与酿酒酵母作为猎物生物的强大遗传学相结合。（iii）他们有两阶段的生活方式。它们在全培养基中腐生生长，但在饥饿时转变为掠食性生活方式。这将允许从代谢基因中解剖毒力基因。（iv）酵母菌属可能是有用的生物防治生物，可用于农业，以减少用于控制植物病原真菌的杀菌剂的用量。基于我们之前广泛的工作，我们在这个项目中提出了四个工作包：(i)进一步表征无毒的KSS1缺失菌株，以回答以下问题：在KSS1突变体中是否有任何猎物细胞的感知？也就是说，捕食者酵母kss1细胞是否仍然朝着猎物生长？kss1细胞是否能够形成穿透钉？互补菌株似乎比野生型菌株更强，即毒性更强。因此，我们将分析KSS1的表达水平是否影响毒力？（ii）从捕食者的角度研究捕食过程，特别是通过解剖极化形态发生和肌动蛋白细胞骨架来监测导致穿透钉形成的事件。我们预计肌动蛋白极化到穿透钉的尖端。然而，我们最感兴趣的是，是否有肌动蛋白环形成在穿透钉的底部，进一步，如果在穿透钉形成和细胞核进入穿透钉的过程中有核分裂的要求。（iii）从被捕食细胞的角度描述捕食过程。这将回答S. cerevisiae猎物细胞的质膜是否（或多长时间）在捕食过程中保持完整的关键问题。在这里，我们还将使用荧光显微镜分析猎物细胞区室的亚细胞变化，例如液泡和线粒体。（iv）分析S. schoenii中mapk信号级联的其他组分，特别是MAP激酶FUS3和下游转录因子STE12。这将回答一个关键问题，即捕食酵母是否以及如何在捕食和交配之间进行优先排序。

Our work aims at the molecular analysis of predation in Saccharomycopsis schoenii.Saccharomycopsis species have strong potential because:(i) they can be easily cultivated, are haploid (our unpublished results), are either homothallic or heterothallic and are amenable to forward and reverse genetics.(ii) they show a unique property of necrotrophic mycoparasitism within Saccharomycetales that provides a simple model to study host-pathogen interactions, e.g. in combination with the powerful genetics of S. cerevisiae as a prey organism.(iii) they have a biphasic life-style. They grow saprophytic in full media but switch to a predatory life-style upon starvation. This will allow dissecting virulence genes from metabolic genes.(iv) Saccharomycopsis species may be useful as biocontrol organisms that could be employed in agriculture to reduce the amount of fungicides used to control plant pathogenic fungi. We propose four work packages within this project based on our extensive previous work:(i) The further characterization of the avirulent KSS1 deletion strain in order to answer the following questions:Is there any sensing of prey cells in the kss1 mutant? That is, do predator yeast kss1 cells still grow towards their prey? Are kss1 cells at all able to form penetration pegs? The complementation strain appeared to be better i.e. more virulent than the wildtype strain. Therefore, we will analyze if the expression level of KSS1 influences virulence?(ii) Study the predation process from the predator’s side, particularly by dissecting polarized morphogenesis and the actin cytoskeleton to monitor events that lead to penetration peg formation. We expect actin polarization to the tip of the penetration peg. Yet, of premier interest to us is if there is actin ring formation at the base of the penetration peg and further if there is a requirement of a nuclear division during penetration peg formation and the entry of a nucleus in the penetration peg. (iii) Characterization of the predation process from the viewpoint of the prey cell. This shall answer the key question whether (or for how long) the plasma membrane of the S. cerevisiae prey cells stays intact during predation. Here we will also analyse subcellular changes in prey cell compartments, e.g. the vacuole and the mitochondria using fluorescence microscopy.(iv) Analysis of additional components of the MAPK-signaling cascade in S. schoenii, particularly the MAP kinase FUS3 and the downstream transcription factor STE12. This shall answer the key question if and how predator yeasts prioritize between predation and mating.