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

Authors: CULVER, JAMES - University Of Maryland; VALLAR, MEINHART - University Of Maryland; Burchard, Erik; KAMENS, SOPHIE - University Of Maryland; LAIR, SEBASTIEN - University Of Maryland; QI, YIPING - University Of Maryland; Collum, Tamara; Dardick, Christopher; EL-MOHTAR, CHOAA - University Of Florida; DAWSON, WILLIAM - University Of Florida; Rogers, Elizabeth

Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2025
Publication Date: 4/10/2025
Citation: Culver, J., Vallar, M., Burchard, E.A., Kamens, S., Lair, S., Qi, Y., Collum, T.D., Dardick, C.D., El-Mohtar, C., Dawson, W., Rogers, E.E. 2025. Citrus phloem specific transcriptional profiling through the development of a citrus tristeza virus expressed translating ribosome affinity purification system. Plant Methods. 21(49). https://doi.org/10.1186/s13007-025-01368-7.
DOI: https://doi.org/10.1186/s13007-025-01368-7

Interpretive Summary: Citrus production in the US is under significant threat from a disease called Huanglongbing (HLB), commonly referred to as Citrus Greening. The causal bacteria is spread by small flying insects (pysllids) and causes severe growth defects and ultimately tree death. Despite decades of intensive research efforts, strategies to combat or prevent HLB are almost non-existent. A significant challenge has been understanding the interactions between the bacteria and the tree host within the infected tissues. The HLB bacterium resides within the tree vascular tissues, which move water and nutrients throughout the plant. These tissues are not readily accessible as they are buried inside the trunk, leaves, and stems. We previously applied a method to isolate plant responses within the vasculature called Translating Ribosome Affinity Purification (TRAP). A limitation of TRAP is that it requires inserting foreign genes into plants which can be time consuming and difficult, especially in tree crops like citrus. In this study, we have modified the TRAP method so that it no longer requires the insertion of genes into the trees. Instead, a disarmed virus that only infects citrus vascular tissues is used to express the TRAP system in the target location. This method allows for the rapid isolation and analysis of citrus responses to HLB and will be useful for better understanding this devastating disease and finding solutions for growers to combat it.

Technical 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.