CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), Centre for Excellence in Molecular Plant Sciences (CEMPS), Shanghai Institute of Plant Physiology and Ecology (SIPPE), Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, China.
New Phytol. 2022 Dec;236(5):1988-1998. doi: 10.1111/nph.18504. Epub 2022 Oct 7.
Visualization of root colonization by arbuscular mycorrhizal fungi (AMF) is the most elementary experiment in the field of mycorrhizal symbiosis. The most widely used approach for evaluating levels of AMF colonization is staining with trypan blue or ink, which is scored using the time-consuming grid intersection method. Here we demonstrate the use of an anthocyanin-based visual marker system for visualizing AMF colonization of Medicago truncatula roots. Expression of MtLAP1, a transcription factor which regulates the production of anthocyanins, from the AMF-induced Kunitz Protease Inhibitor 106 promoter, allowed the visualization of arbuscules in live plant tissues without microscopy or staining. This marker system allowed straightforward qualitative evaluation of the ram1, vpy and dmi3 AMF phenotypes using Agrobacterium rhizogenes hairy-root transformation. For the strigolactone biosynthesis mutant carotenoid cleavage dioxygenase 8a and a novel mutant scooby, which show quantitative AMF symbiotic phenotypes, the amount of anthocyanins in the roots estimated by spectrophotometry correlated very well with colonization levels estimated by staining and scoring using the grid intersection method. The LAP1-based marker system therefore provides a highly efficient approach for mutant screening and monitoring of AMF colonization in live tissues by eye, or for quantitative assessment using a simple and quick photometric assay.
菌根真菌（AMF）定殖的可视化是菌根共生领域最基本的实验。评估 AMF 定殖水平最广泛使用的方法是用台盼蓝或墨水染色，然后使用耗时的网格交叉法进行评分。在这里，我们展示了一种基于花青素的可视化标记系统，用于可视化紫花苜蓿根系中的 AMF 定殖。来自 AMF 诱导的 Kunitz 蛋白酶抑制剂 106 启动子的 MtLAP1 转录因子的表达，调控花青素的产生，使得在没有显微镜或染色的情况下，可以在活体植物组织中可视化丛枝。该标记系统允许使用根癌农杆菌毛状根转化，对 ram1、vpy 和 dmi3 AMF 表型进行简单的定性评估。对于生物合成突变体胡萝卜素双加氧酶 8a 和新型突变体 scooby，它们表现出定量的 AMF 共生表型，分光光度法估计的根中花青素含量与染色和网格交叉法评分估计的定殖水平非常吻合。因此，基于 LAP1 的标记系统为在活体组织中通过肉眼进行突变体筛选和 AMF 定殖监测，或使用简单快速的分光光度法进行定量评估提供了一种高效的方法。
