Abstract
BackgroundAgrobacterium 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.ResultsHere, 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.ConclusionsAnthocyanin 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.
Highlights
Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis
To overcome the drawbacks of traditional methods for selection transgenic hairy roots, here, we developed a novel reporter gene AtMyb75/PAP1 that can be used in the study of rhizobia-legume symbiosis without interfering with nitrogen-fixing nodule development
AtMyb75/PAP1 was ectopically expressed in hairy roots-mediated by A. rhizogenes, that induced the purple/red colored anthocyanin accumulation in the legumes soybean (Glycine max (L.) Merr.) with purple hypocotyl, L. japonicus, L. corniculatus, and M. truncatula
Summary
Agrobacterium rhizogenes-mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. In the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required. Fan et al Plant Methods (2020) 16:94 of transgenic roots based on reporter genes, the β-glucuronidase (GUS) activity or fluorescent proteins (such as GFP, YFP, RFP, CFP, etc.) were the most widely utilized [1, 5,6,7,8,9,10,11,12]. GUS staining assay is destructive to plant tissues, and an expensive chemical substrate (X-Gluc) is required. A prominent feature of fluorescent proteins is a nondestructive and visual reporter without a requirement of an additional substrate. It is not a convenient operation to integrate four Neonothopanus nambi bioluminescence genes: nnluz (luciferase), nnhisps (hispidin synthase), nnh3h (hispidin-3-hydroxylase) and nncph (caffeoyl pyruvate hydrolase) into plants
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