Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required. Here, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene AtMyb75 in Arabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by A. rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max (L.) Merr.) and two model legume species, Lotus japonicas and Medicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of AtMyb75 as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation Rfg1 gene in the soybean PI377578 (Nod-) inoculated with Sinorhizobium fredii USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin. Anthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.

中文翻译：

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花青素，一种新颖且用户友好的报告基因，可在根瘤菌-豆科植物共生研究中方便、无损、低成本、直接视觉选择转基因毛状根。


发根农杆菌介导的毛状根转化为研究涉及根瘤菌-豆科植物共生的植物基因的功能提供了有力的工具。然而，传统的基于报告基因的转基因毛状根鉴定方法需要昂贵的化学底物或设备。在这里，我们报道了一种新颖、低成本、稳健的报告基因，可通过肉眼方便、非破坏性、直接视觉选择转基因毛状根，可用于根瘤菌-豆科植物共生的研究。拟南芥中的报告基因 AtMyb75 编码 R2R3 型 MYB 转录因子，在发根农杆菌介导的毛状根中异位表达，从而诱导大豆 (Glycine max (L.) Merr.) 等农作物中紫色/红色花青素的积累。 ）和两种模式豆科植物，Lotus japonicas 和 Medicago truncatula。含有花青素的转基因豆科植物毛状根可以与根瘤菌建立有效的共生关系。我们还通过对接种了中华根瘤菌 USDA193 的大豆 PI377578 (Nod-) 中的大豆抗结瘤 Rfg1 基因进行 CRISPR/Cas9 靶向诱变，证明了 AtMyb75 作为报告基因的可靠性。无一例外，在含有花青素的紫色转基因毛状根上形成了成熟的固氮根。花青素是一种可靠、用户友好、方便、无损、低成本、直接可视化的报告基因，用于研究共生固氮根瘤的发育，可广泛应用于广大豆科植物。