Regulation of reactive oxygen species metabolism in early infection stages of Fusarium graminearum on wheat

禾谷镰刀菌（Fusarium graminearum）是一种坏死营养型丝状子囊菌，是世界上最重要的植物病原菌之一。它对所有谷物都是致病性的，通过霉菌毒素的积累导致作物损失以及食物和饲料中毒。禾谷镰刀菌形成感染垫（IC），这是一种特殊的复杂感染结构，可以穿透代表自然感染部位的小麦花叶表面。利用激光解剖显微镜从小麦叶片上的侵染垫中分离出了附生生长的匍匐菌丝。任一结构的转录谱显示了一个大的基因组，特别是在RH和IC上调，分别。这些基因的一个子集编码分泌酶，其在活性氧（ROS）的代谢中发挥作用，活性氧是许多氧化过程的副产物。ROS具有不同的重要功能，即基因调控、细胞信号传导和发育、参与细胞凋亡和病原体防御。在坏死性真菌中，它可能作为一种侵略性的代理人。它们可以在细胞内和细胞外运作。本研究组最近的研究使我们假设ROS的产生和分泌是F. graminearum到小麦。在早期感染阶段的ROS平衡的严格监管似乎是至关重要的结果的interaction.In拟议的项目中，我们将集中在35个分泌型ROS相关酶（SRE），在感染垫的发展过程中上调。我们将删除的基因开始与低遗传冗余的功能相关的基因，从而专注于ROS解毒。接下来，功能组的基因将被破坏;从最高表达的基因开始。突变体将在广泛的表型筛选中进行分析，包括ROS代谢、感染垫发育和渗透、小麦和玉米的毒力测定以及DON测量。将使用诱导型表达系统补充具有感兴趣表型的突变体。这使得小麦花叶表面上的原位互补成为可能。在F.本研究将利用我们实验室最近开发的适用于丝状真菌的比率过氧化氢传感器蛋白质HyPer，对小麦中的禾谷镰刀菌进行可视化研究。禾谷镰刀菌的毒力研究在我们的实验室，和拟议的博士生将由技术援助，学士和硕士生，和申请人的支持。转录组学研究、正向遗传工具和使用比率传感器蛋白的生命细胞成像的组合代表了鉴定对于ROS稳态、复杂感染结构的发展以及植物的成功渗透和定殖至关重要的蛋白质的有力工具。

Fusarium graminearum is a necrotrophic, filamentous ascomycete and one of the most important phytopathogens worldwide. It is pathogenic to all cereals, causing crop losses and food and feed poising by accumulation of mycotoxins. Fusarium graminearum forms infection cushions (IC), specialized complex infection structures to penetrate the surface of wheat floral leafs which represent the natural infection site. We separated epiphytically growing runner hyphae (RH) from infection cushions on colonized wheat floral leaves by laser dissecting microscopy. Transcriptional profiling of either structure revealed a large set of genes specifically up regulated in RH and IC, respectively. A subset of these genes encodes secreted enzymes, which play a role in the metabolism of reactive oxygen species (ROS), byproducts of many oxidative processes. ROS have different important functions, i.e. gene regulation, cell signaling and development, involvement in apoptosis, and pathogen defense. In necrotophic fungi, it may act as an aggressive agent. They can operate intra- and extracellular. Recent investigations in our group led us to the assumption that production and secretion of ROS represents an important factor for virulence of F. graminearum to wheat. Tight regulation of ROS-equilibrium during early infection stages seems to be crucial for the outcome of the interaction.In the proposed project we will concentrate on 35 secreted ROS-related enzymes (SREs), up regulated during infection cushion development. We will delete the genes starting with functionally related genes with low genetic redundancy, thereby focusing on ROS detoxification. Next, genes of functional groups will be disrupted; starting with the highest expressed ones. Mutants will be analyzed in a broad scale phenotypic screening including ROS metabolism, infection cushion development and penetration, virulence assays on wheat and maize, and DON-measurements. Mutants with interesting phenotypes will be complemented using an inducible expression system. This enables an in-situ complementation on the surface of wheat flower leafs. ROS-dynamics during the initial infection of F. graminearum in wheat will be visualized using the ratiometric hydrogen peroxide sensor protein HyPer, recently adapted for filamentous fungi in our lab.The proposed project is part of the F. graminearum virulence studies in our lab, and the proposed PhD student will be supported by technical assistances, bachelor and master students, and the applicant. The combination of transcriptomic studies, forward genetic tools and life cell imaging using ratiometric sensor proteins represents a powerful tool to identify proteins that are essential for ROS-homeostasis, development of complex infection structures, and for successful penetration and colonization of the plant.