Table_1_A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules.DOCX

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus, for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia interactions.

豆科植物与定殖于特化根瘤（root nodule）内的根瘤菌（rhizobia）形成固氮共生关系，从而获得生长益处。根瘤的形成起始于从头器官发生（de novo organogenesis），且需根瘤菌对这些发育中的侧生根器官进行协同侵染。细菌侵染与根瘤器官发生均涉及细胞周期激活，而生长素与细胞分裂素的调控在该过程中被紧密整合。为在结瘤过程中以细胞分辨率解析激素动态与细胞分裂模式，亟需适用于多细胞组织成像的高灵敏度、特异性传感器。本研究报道了一套模块化工具包，已在模式豆科植物百脉根（Lotus japonicus）中完成优化，可应用于豆科植物根系及根瘤。该工具包包含针对生长素与细胞分裂素的合成转录报告基因、生长素积累传感器，以及经荧光与明场显微镜优化的细胞周期进程标记物。所开发的载体可利用GoldenGate克隆系统实现多元件的高效一步组装。结合荧光兼容的组织透明化方法使用时，这些报告基因可提升成像深度，并便于在豆科植物根系中开展荧光检测。我们还评估了动态向重力性根系响应在改变生长素积累的时机与位置，以及根瘤萌发中的作用。研究表明，改变根系内的生长素分布，可使根瘤优先在弯曲处的外侧区域萌发，该区域对应生长素信号传导能力较高的位点。本研究展示的工具与方法，为比较突变体研究以及更全面地解析豆科植物-根瘤菌互作机制提供了新的可能。