A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus
The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses...
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MDPI AG
2022-07-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/11/13/1765 |
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| author | Weiwei Chen Chaolin Huang Chenmeng Luo Yongshan Zhang Bin Zhang Zhengqing Xie Mengyuan Hao Hua Ling Gangqiang Cao Baoming Tian Fang Wei Gongyao Shi |
| author_facet | Weiwei Chen Chaolin Huang Chenmeng Luo Yongshan Zhang Bin Zhang Zhengqing Xie Mengyuan Hao Hua Ling Gangqiang Cao Baoming Tian Fang Wei Gongyao Shi |
| author_sort | Weiwei Chen |
| collection | DOAJ |
| description | The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na<sup>+</sup> from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research. |
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| id | doaj.art-375ba6bb4cb947e18dce66399417b0a7 |
| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-09T03:57:19Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-375ba6bb4cb947e18dce66399417b0a72023-12-03T14:18:05ZengMDPI AGPlants2223-77472022-07-011113176510.3390/plants11131765A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic VirusWeiwei Chen0Chaolin Huang1Chenmeng Luo2Yongshan Zhang3Bin Zhang4Zhengqing Xie5Mengyuan Hao6Hua Ling7Gangqiang Cao8Baoming Tian9Fang Wei10Gongyao Shi11Zhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaHenan International Joint Laboratory of Crop Gene Resources and Improvements, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaZhengzhou Research Base, State Key Laboratory of Cotton Biology, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, ChinaThe difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na<sup>+</sup> from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.https://www.mdpi.com/2223-7747/11/13/1765cottonbarley stripe mosaic virus (BSMV)organelle markersubcellular localizationprotein–protein interactionsCRISPR editing |
| spellingShingle | Weiwei Chen Chaolin Huang Chenmeng Luo Yongshan Zhang Bin Zhang Zhengqing Xie Mengyuan Hao Hua Ling Gangqiang Cao Baoming Tian Fang Wei Gongyao Shi A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus Plants cotton barley stripe mosaic virus (BSMV) organelle marker subcellular localization protein–protein interactions CRISPR editing |
| title | A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus |
| title_full | A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus |
| title_fullStr | A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus |
| title_full_unstemmed | A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus |
| title_short | A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus |
| title_sort | new method for rapid subcellular localization and gene function analysis in cotton based on barley stripe mosaic virus |
| topic | cotton barley stripe mosaic virus (BSMV) organelle marker subcellular localization protein–protein interactions CRISPR editing |
| url | https://www.mdpi.com/2223-7747/11/13/1765 |
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