Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system

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

doi:10.1186/s13007-025-01368-7

Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system

Plant Methods. 2025 Apr 10;21(1):49. doi: 10.1186/s13007-025-01368-7.

Authors

Affiliations

¹ Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA. jculver@umd.edu.
² Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA. jculver@umd.edu.
³ Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD, USA.
⁴ USDA, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV, USA.
⁵ Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
⁶ Department of Plant Pathology, Citrus Research and Education Center, University of Florida, Gainesville, FL, USA.
⁷ USDA, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Frederick, MD, USA.

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.

Keywords: Citrus translatome analysis; Phloem gene expression; Translating ribosome affinity purification (TRAP); Virus vector.

Abstract

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