Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system
Abstract Background The analysis of translationally active mRNAs, or translatome, is a useful approach for monitoring cellular and plant physiological responses. One such method is the translating ribosome affinity purification (TRAP) system, which utilizes tagged ribosomal proteins to isolate ribos...
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BMC
2025-04-01
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| Series: | Plant Methods |
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| Online Access: | https://doi.org/10.1186/s13007-025-01368-7 |
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| author | James N. Culver Meinhart Vallar Erik Burchard Sophie Kamens Sebastien Lair Yiping Qi Tamara D. Collum Christopher Dardick Choaa A. El-Mohtar Elizabeth E. Rogers |
| author_facet | James N. Culver Meinhart Vallar Erik Burchard Sophie Kamens Sebastien Lair Yiping Qi Tamara D. Collum Christopher Dardick Choaa A. El-Mohtar Elizabeth E. Rogers |
| author_sort | James N. Culver |
| collection | DOAJ |
| description | Abstract Background The analysis of translationally active mRNAs, or translatome, is a useful approach for monitoring cellular and plant physiological responses. One such method is the translating ribosome affinity purification (TRAP) system, which utilizes tagged ribosomal proteins to isolate ribosome-associated transcripts. This approach enables spatial and temporal gene expression analysis by driving the expression of tagged ribosomal proteins with tissue- or development-specific promoters. In plants, TRAP has enhanced our understanding of physiological responses to various biotic and abiotic factors. However, its utility is hampered by the necessity to generate transgenic plants expressing the tagged ribosomal protein, making this approach particularly challenging in perennial crops such as citrus. Results This study involved the construction of a citrus tristeza virus (CTV) vector to express an immuno-tagged ribosome protein (CTV-hfRPL18). CTV, limited to the phloem, has been used for expressing marker and therapeutic sequences, making it suitable for analyzing citrus vascular tissue responses, including those related to huanglongbing disease. CTV-hfRPL18 successfully expressed a clementine-derived hfRPL18 peptide, and polysome purifications demonstrated enrichment for the hfRPL18 peptide. Subsequent translatome isolations from infected Nicotiana benthamiana and Citrus macrophylla showed enrichment for phloem-associated genes. Conclusion The CTV-hfRPL18 vector offers a transgene-free and rapid system for TRAP expression and translatome analysis of phloem tissues within citrus. |
| format | Article |
| id | doaj-art-eb7fce40d9a743858b9f0bfaa8307cb7 |
| institution | DOAJ |
| issn | 1746-4811 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj-art-eb7fce40d9a743858b9f0bfaa8307cb72025-08-20T03:10:07ZengBMCPlant Methods1746-48112025-04-0121111010.1186/s13007-025-01368-7Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification systemJames N. Culver0Meinhart Vallar1Erik Burchard2Sophie Kamens3Sebastien Lair4Yiping Qi5Tamara D. Collum6Christopher Dardick7Choaa A. El-Mohtar8Elizabeth E. Rogers9Institute for Bioscience and Biotechnology Research, University of MarylandInstitute for Bioscience and Biotechnology Research, University of MarylandUSDA, Agricultural Research Service, Appalachian Fruit Research StationInstitute for Bioscience and Biotechnology Research, University of MarylandInstitute for Bioscience and Biotechnology Research, University of MarylandDepartment of Plant Science and Landscape Architecture, University of MarylandUSDA, Agricultural Research Service, Appalachian Fruit Research StationUSDA, Agricultural Research Service, Appalachian Fruit Research StationDepartment of Plant Pathology, Citrus Research and Education Center, University of FloridaUSDA, Agricultural Research Service, Foreign Disease-Weed Science Research UnitAbstract Background The analysis of translationally active mRNAs, or translatome, is a useful approach for monitoring cellular and plant physiological responses. One such method is the translating ribosome affinity purification (TRAP) system, which utilizes tagged ribosomal proteins to isolate ribosome-associated transcripts. This approach enables spatial and temporal gene expression analysis by driving the expression of tagged ribosomal proteins with tissue- or development-specific promoters. In plants, TRAP has enhanced our understanding of physiological responses to various biotic and abiotic factors. However, its utility is hampered by the necessity to generate transgenic plants expressing the tagged ribosomal protein, making this approach particularly challenging in perennial crops such as citrus. Results This study involved the construction of a citrus tristeza virus (CTV) vector to express an immuno-tagged ribosome protein (CTV-hfRPL18). CTV, limited to the phloem, has been used for expressing marker and therapeutic sequences, making it suitable for analyzing citrus vascular tissue responses, including those related to huanglongbing disease. CTV-hfRPL18 successfully expressed a clementine-derived hfRPL18 peptide, and polysome purifications demonstrated enrichment for the hfRPL18 peptide. Subsequent translatome isolations from infected Nicotiana benthamiana and Citrus macrophylla showed enrichment for phloem-associated genes. Conclusion The CTV-hfRPL18 vector offers a transgene-free and rapid system for TRAP expression and translatome analysis of phloem tissues within citrus.https://doi.org/10.1186/s13007-025-01368-7Virus vectorPhloem gene expressionCitrus translatome analysisTranslating ribosome affinity purification (TRAP) |
| spellingShingle | James N. Culver Meinhart Vallar Erik Burchard Sophie Kamens Sebastien Lair Yiping Qi Tamara D. Collum Christopher Dardick Choaa A. El-Mohtar Elizabeth E. Rogers Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system Plant Methods Virus vector Phloem gene expression Citrus translatome analysis Translating ribosome affinity purification (TRAP) |
| title | Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| title_full | Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| title_fullStr | Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| title_full_unstemmed | Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| title_short | Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| title_sort | citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system |
| topic | Virus vector Phloem gene expression Citrus translatome analysis Translating ribosome affinity purification (TRAP) |
| url | https://doi.org/10.1186/s13007-025-01368-7 |
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