Zhang Shiqi, Jurgensen Lucas, Harrison Maria J
Boyce Thompson Institute, 533 Tower Road, Ithaca, NY, USA.
Bio Protoc. 2025 Jan 20;15(2):e5158. doi: 10.21769/BioProtoc.5158.
Arbuscular mycorrhizal (AM) fungi engage in symbiotic relationships with plants, influencing their phosphate (Pi) uptake pathways, metabolism, and root cell physiology. Despite the significant role of Pi, its distribution and response dynamics in mycorrhizal roots remain largely unexplored. While traditional techniques for Pi measurement have shed some light on this, real-time cellular-level monitoring has been a challenge. With the evolution of quantitative imaging with confocal microscopy, particularly the use of genetically encoded fluorescent sensors, live imaging of intracellular Pi concentrations is now achievable. Among these sensors, fluorescence resonance energy transfer (FRET)-based biosensors stand out for their accuracy. In this study, we employ the Pi-specific biosensor (cpFLIPPi-5.3m) targeted to the cytosol or plastids of plants, enabling us to monitor intracellular Pi dynamics during AM symbiosis. A complementary control sensor, cpFLIPPi-Null, is introduced to monitor non-Pi-specific changes. Leveraging a semi-automated ImageJ macro for sensitized FRET analysis, this method provides a precise and efficient way to determine relative intracellular Pi levels at the level of individual cells or organelles. Key features • This protocol describes the use of FRET biosensors for in vivo visualization of spatiotemporal phosphate levels with cellular and subcellular resolution in . • An optimized growth system can allow tracing of Pi transfer between AM fungi and host root. New Phytol (2022), DOI: 10.1111/nph.18081.
丛枝菌根（AM）真菌与植物形成共生关系，影响植物的磷（Pi）吸收途径、代谢和根细胞生理学。尽管磷起着重要作用，但其在菌根根中的分布和响应动态仍 largely unexplored。虽然传统的磷测量技术对此有所揭示，但实时细胞水平监测一直是一项挑战。随着共聚焦显微镜定量成像技术的发展，特别是基因编码荧光传感器的使用，现在可以实现细胞内磷浓度的实时成像。在这些传感器中，基于荧光共振能量转移（FRET）的生物传感器因其准确性而脱颖而出。在本研究中，我们使用针对植物细胞质或质体的磷特异性生物传感器（cpFLIPPi - 5.3m），使我们能够监测AM共生过程中的细胞内磷动态。引入了一个互补的对照传感器cpFLIPPi - Null来监测非磷特异性变化。利用半自动ImageJ宏进行敏化FRET分析，该方法提供了一种精确有效的方法来确定单个细胞或细胞器水平上的相对细胞内磷水平。关键特性 • 本方案描述了使用FRET生物传感器在体内以细胞和亚细胞分辨率可视化时空磷水平。 • 优化的生长系统可以追踪AM真菌与宿主根之间的磷转移。《新植物学家》（2022），DOI：10.1111/nph.18081 。 （注：“largely unexplored”直译为“很大程度上未被探索”，这里意译为“仍不清楚”更通顺些。）
