Data_Sheet_3_Broad Substrate-Specific Phosphorylation Events Are Associated With the Initial Stage of Plant Cell Wall Recognition in Neurospora crassa.XLSX

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

真菌植物细胞壁降解过程受复杂调控机制支配，使得该类生物能够针对可利用底物以高度特异性调整自身代谢程序。尽管已知典型植物细胞壁单糖及二糖的摄取会诱导特异性转录与翻译应答，但与早期信号接收及转导相关的过程仍在很大程度上未被阐明。信号传递的一种快速且可逆的方式为翻译后蛋白质修饰（如磷酸化修饰），此类修饰可启动真菌代谢对新环境的快速适应。为阐明粗糙脉孢菌（Neurospora crassa）初始底物识别阶段的变化如何影响全局磷酸化谱，研究团队在使用数种植物细胞壁相关单糖及二糖进行短时长（2分钟）诱导后，开展了磷酸化蛋白质组学（phospho-proteomics）分析。基于串联质谱（MS/MS）的肽段分析揭示了大规模底物特异性蛋白质磷酸化与去磷酸化事件。利用串联质谱鉴定得到的蛋白质，构建了蛋白质-蛋白质相互作用（PPI）网络。大量激酶、磷酸酶及转录因子的磷酸化水平变化表明，诸多已知信号通路均参与其中，包括昼夜节律应答、双组分调控系统、丝裂原活化蛋白激酶（MAP kinases）通路，以及环腺苷酸依赖型与异三聚体G蛋白通路。作为环腺苷酸通路关键组分的腺苷酸环化酶，被鉴定为碳源特异性差异蛋白质相互作用的潜在核心节点。此外，研究共鉴定出4种磷酸化F盒蛋白，其中Fbx-19与Fbx-22这两种蛋白被发现参与碳分解代谢物阻遏应答过程。综上，本研究结果为真菌响应环境信号的迄今尚未被充分解析的阶段提供了前所未有的详尽认知，并有助于更好地阐明植物细胞壁降解过程中感官感知与信号转导的分子机制。