The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

切叶蚁真菌花园生态系统是一个自然进化的植物生物量高效降解模型系统。由于系统的动态代谢和空间复杂性，共生真菌Leucoagaricus gogylophorus介导的降解过程很难表征。在此，我们对Atta cephalotes真菌花园12 µm 厚的相邻部分进行了微尺度成像，并应用代谢组信息的蛋白质组成像方法来绘制木质素降解图。这种方法结合了两种空间多组学质谱模式，使我们能够可视化整个真菌花园中的共定位代谢物和蛋白质。代谢物的空间分布揭示了木质素相关产物的积累，概述了形态独特的木质素微生境。这些微生境的宏蛋白质组学分析揭示了碳水化合物降解酶，表明真菌在木质纤维素分解中发挥着重要作用。代谢组信息蛋白质组成像数据的整合提供了潜在生物途径的全面视图，有助于我们了解微米级环境中植物物质降解中的代谢真菌途径。