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
Abstract 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 expe...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2020-07-01
|
| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-020-00638-w |
| _version_ | 1818533275065384960 |
|---|---|
| author | Yinglun Fan Xiuyuan Wang Haiyun Li Shuang Liu Liangshen Jin Yanyan Lyu Mengdi Shi Sirui Liu Xinyue Yang Shanhua Lyu |
| author_facet | Yinglun Fan Xiuyuan Wang Haiyun Li Shuang Liu Liangshen Jin Yanyan Lyu Mengdi Shi Sirui Liu Xinyue Yang Shanhua Lyu |
| author_sort | Yinglun Fan |
| collection | DOAJ |
| description | Abstract 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 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. 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. |
| first_indexed | 2024-12-11T17:56:32Z |
| format | Article |
| id | doaj.art-c6e8289faf9243e980a12c64e4485c71 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-11T17:56:32Z |
| publishDate | 2020-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-c6e8289faf9243e980a12c64e4485c712022-12-22T00:56:03ZengBMCPlant Methods1746-48112020-07-011611810.1186/s13007-020-00638-wAnthocyanin, 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 symbiosisYinglun Fan0Xiuyuan Wang1Haiyun Li2Shuang Liu3Liangshen Jin4Yanyan Lyu5Mengdi Shi6Sirui Liu7Xinyue Yang8Shanhua Lyu9College of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityCollege of Agriculture, Liaocheng UniversityAbstract 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 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. 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.http://link.springer.com/article/10.1186/s13007-020-00638-wReporter geneAnthocyaninHairy rootRhizobia-legume symbiosisSoybean (Glycine max (L.) Merr.)Lotus japonicus |
| spellingShingle | Yinglun Fan Xiuyuan Wang Haiyun Li Shuang Liu Liangshen Jin Yanyan Lyu Mengdi Shi Sirui Liu Xinyue Yang Shanhua Lyu 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 Plant Methods Reporter gene Anthocyanin Hairy root Rhizobia-legume symbiosis Soybean (Glycine max (L.) Merr.) Lotus japonicus |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Reporter gene Anthocyanin Hairy root Rhizobia-legume symbiosis Soybean (Glycine max (L.) Merr.) Lotus japonicus |
| url | http://link.springer.com/article/10.1186/s13007-020-00638-w |
| work_keys_str_mv | AT yinglunfan anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT xiuyuanwang anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT haiyunli anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT shuangliu anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT liangshenjin anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT yanyanlyu anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT mengdishi anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT siruiliu anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT xinyueyang anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis AT shanhualyu anthocyaninanovelanduserfriendlyreporterforconvenientnondestructivelowcostdirectlyvisualselectionoftransgenichairyrootsinthestudyofrhizobialegumesymbiosis |