Genetically encoded Ca²⁺ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca²⁺ signaling in fungi, ranging from documenting Ca²⁺ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca²⁺ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca²⁺ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca²⁺ indicator. Wild-type and three Ca²⁺ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

基因编码的 Ca ²⁺指示剂 (GECI) 能够长期监测第二信使的细胞和亚细胞动态，以响应环境和发育线索，而无需依赖外源染料。GECI 的持续开发和优化，结合基因操作的进步，为研究真菌中 Ca ²⁺信号传导机制提供了新的机会，从记录不同条件和遗传背景下的Ca ²⁺特征到评估 Ca ²⁺如何变化特征影响钙结合蛋白和随后的细胞变化。在这里，我们试图表达多色（绿色、黄色、蓝色、青色和红色）循环排列的基于荧光蛋白 (FP) 的 Ca²⁺指标通过在多个真菌启动子驱动的尖孢镰孢，˚F。禾本科和粗糙脉孢菌。成功表达了几种变体，其中由稻瘟病菌核糖体蛋白 27驱动的 GCaMP5G和轮枝杆菌延伸因子-1α 基因启动子最适合F。禾本科和F . 分别为尖孢菌。将表达 GCaMP5G 的转化体与表达 YC3.60（一种比例 Cameleon Ca ²⁺指示剂）的转化体进行比较。野生型和三个 Ca ²⁺信号突变体˚F。与表达 YC3.60 的菌株相比，表达 GCaMP5G 的禾本科表现出改善的信噪比和增加的时间和空间分辨率，并且也更适合涉及多个 FP 的研究。