A novel role for polysaccharide monooxygenases in signaling, chemotropic interactions and cell fusion

据估计，世界上多达四分之一的生物量是真菌来源，包括大约600万种。为了相互作用并对环境线索做出反应，这些物种通过化学语言进行交流和反应。在丝状子囊菌真菌中，萌发的无性孢子和菌丝通过交流和融合形成一个相互连接的多核菌丝网络。在自然界中，丝状真菌在菌丝网络中对植物和动物物质的分解、养分循环和养分运输发挥着重要作用，包括作为共生体和病原体与植物的联系。本项目定义了一类被称为多糖单加氧酶（PMOs）的氧化酶在菌丝网络形成过程中的新生物学功能，这些酶在丝状真菌体细胞融合过程中的信号传导、趋化相互作用和细胞壁重塑中发挥作用。以前，这类酶只与细胞外降解能力有关，为这类蛋白质的细胞功能提供了范式转变。铜依赖性PMOs在细胞信号传导中的作用的发现，以及这些酶的新底物的表征，将极大地影响这一研究领域；在人类和植物病原体中发现了编码PMOs的基因，也可能涉及共生关系。该项目汇集了细胞信号、细胞生物学和遗传学方面的专业知识，并在PMOs的生物化学和结构生物学方面拥有深厚的专业知识，为本科生、研究生和博士后提供了良好的培训机会。与教育科学组织合作，将利用丝状真菌粗神经孢子菌来识别和表征参与细胞-细胞通信和融合的分子参与者，并将形成加州大学伯克利分校为期三周的强化本科课程的基础。在旧金山真菌学学会和湾区真菌学学会提供的展览中，本项目和其他项目的活细胞成像电影将丰富真菌在环境中的作用的教育方面。真菌孢子（或菌落中的菌丝）之间的趋化相互作用启动细胞通信，随后在粘附细胞之间的接触点从细胞生长转变为细胞壁破裂和膜合并。预测的PMO HAM-7在丝状真菌的细胞壁完整性（CWI） MAP激酶信号通路中的趋化相互作用和功能是必需的。缺乏ham-7的菌株无法进行细胞融合，因此缺乏相互连接的菌丝网络。ham-7基因及其相关基因在真菌的PMOs中形成了一个独特的分支。HAM-7 PMO的底物目前尚不清楚，利用生化方法、结构生物学、细胞生物学和遗传学来确定HAM-7的活性、底物及其在趋化相互作用和细胞融合中的作用是本项目的主要目标。第二种PMO， cwr1，基于同源性，被预测使用几丁质作为底物，在细胞融合过程中从细胞生长到细胞壁分解的转换中起负调节作用。作为一种预测的膜蛋白，cwr3是cwr1在细胞壁分解中发挥作用所必需的，这表明它可能是cwr1酶活性产物的受体。该项目将把多糖单加氧酶的氧化能力与胚芽/菌丝通讯中化学语言的神秘特性联系起来，这些化学语言决定了细胞寻求与自己的同类进行营养融合的行为。本项目所描述的工作将在定义这类酶/蛋白质的新生物学作用方面具有变革性，这些酶/蛋白质可能广泛适用于真核和原核物种，这些物种在其基因组中编码PMOs。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

It has been estimated that up to one quarter of the world's biomass is of fungal origin, comprising approximately six million species. To interact with one another and respond to environmental cues, these species communicate and respond via chemical languages. In filamentous ascomycete fungi, the formation of an interconnected, multinucleate hyphal network is constructed via communication and fusion of germinated asexual spores (germlings) and hyphae. In nature, filamentous fungi play important roles in decomposition of plant and animal matter, nutrient cycling and nutrient transport in the hyphal network, including an association with plants, both as symbionts and as pathogens. This project defines a new biological function during hyphal network formation for a class of oxidative enzymes, termed polysaccharide monooxygenases (PMOs), that play a role during signaling, chemotropic interactions and cell wall remodeling during somatic cell fusion in filamentous fungi. Previously, this class of enzymes has only been associated with extracellular degradative capacities, providing a paradigm shift in the cellular function of this class of proteins. The discovery of a role for copper-dependent PMOs in cell signaling, along with the characterization of new substrates for these enzymes, will greatly impact this field of research; genes encoding PMOs have been found in human and plant pathogens and may also be involved in symbiotic relationships. This project brings expertise on cell signaling, cell biology and genetics with deep expertise in biochemistry and structural biology on PMOs, and provides an excellent training opportunity for undergraduate, graduate students and postdoctoral associates. Workshops will be developed on the use of the filamentous fungus Neurospora crassa to identify and characterize molecular players involved in cell-cell communication and fusion in collaboration with educational science organizations and will form the basis of an intensive three-week undergraduate course at the University of California - Berkeley. Live cell imaging movies from this project and others will enrich educational aspects on the role of fungi in the environment in exhibitions provided by the Mycological Society of San Francisco and the Bay Area Mycological Society. Chemotropic interactions between fungal germlings (or hyphae in a colony), initiates cell communication, followed by a switch from cell growth to cell wall breakdown and membrane merger at the contact point between adhered cells. A role for a predicted PMO, HAM-7, is required for chemotropic interactions and functions through the cell wall integrity (CWI) MAP kinase signaling pathway in filamentous fungi. Strains lacking ham-7 fail to undergo cell fusion and thus lack an interconnected hyphal network. The ham-7 gene and its relatives form a unique clade among PMOs in fungi. The substrate for the HAM-7 PMO is currently unknown and defining the activity of HAM-7, its substrate and role in chemotropic interactions and cell fusion using biochemical methods, structural biology, cell biology and genetics is a major objective of this project. A second PMO, CWR-1, which is predicted to use chitin as a substrate based on homology, functions as a negative regulator of the switch from cell growth to cell wall disassembly during cell fusion. A predicted membrane protein, CWR-3 is required for CWR-1 to function in cell wall disassembly, suggesting it may function as a receptor for the product of the CWR-1 enzymatic activity. This project will link the function of polysaccharide monooxygenases oxidative capacity with the enigmatic properties of chemical languages involved in germling/hyphal communication that dictates the behavior of cells seeking to undergo vegetative fusion with their own kind. The work described in this project will be transformative in defining new biological roles for this class of enzymes/proteins that may be broadly applicable to both eukaryotic and prokaryotic species that encode PMOs in their genomes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.