Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis

Background 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. Results 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 geneAtMyb75inArabidopsis, encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated byA.rhizogenes, which induced purple/red colored anthocyanin accumulation in crop species like soybean (Glycine max(L.) Merr.) and two model legume species,Lotus japonicasandMedicago truncatula. Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability ofAtMyb75as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulationRfg1gene in the soybean PI377578 (Nod-) inoculated withSinorhizobium frediiUSDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin. Conclusions 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.