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 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.

Fig. 1

CTV-hfRPL18 construct. a, Diagram of CTV-hfRPL18 genome showing replacement of the CTV p13 ORF with that of hfRPL18 (red). b and c, Disease symptoms associated with CTV-hfRPL18 infection in N. benthamiana (b) and C. macrophylla (c)

Fig. 2

Stability of CTV-hfRPL18 within infected C. macrophylla trees at 3- and 17-months post-infection. a, Amplified RT-PCR products using primers flanking the hfRPL18 ORF at 3 months post-infection. b, RT-PCR products using both flanking and hfRPL18 specific primers at 17 months post-infection. λPst (marker lane), + (plasmid CTV-hfRPL18), - (RT-PCR of RNA from a healthy tree), CTV (RT-PCR of RNA from CTV empty vector infected tree). Arrows indicate the expected location for full length product for flanking or hfRPL18 specific primers

Fig. 3

hfRPL18 purification and associated RNA extractions. a and b, Detection of hfRPL18 by Western immunoblot at different purification steps in N. benthamiana and C. macrophylla displaying enrichment for the hfRPL18 protein from magnetic bead elution fractions. c, RNA levels obtained from magnetic beads eluted hfRPL18 protein. Bars represent average ± standard deviation (Table S4)

Fig. 4

Gene ontology of C. macrophylla identified phloem associated genes. GO analysis was done using the graphical gene-set enrichment tool ShinyGO (Table S6) [34]

Fig. 5

Phloem expression of CTV-hfRPL18 identified genes in N. benthamiana. A set of six previously identified N. benthamiana phloem-associated mRNAs were assessed for their presence in the CTV-hfRPL18 enriched translatome [12]. PCR amplification for each gene show all expressed within the CTV-hfRPL18 translatome with four being enriched in comparison to extracted total RNA

Fig. 6

Promoter expression analysis of selected Citrus x clementina CTV-hfRPL18 phloem associated mRNAs. Promoter sequences from four genes enriched in the CTV-hfRPL18 translatome were used to drive GUS expression in Arabidopsis (Table S3). GUS stained leaves from representative plants transformed with promoters from a, Ciclev10030495m callose synthase-like 7; b, Ciclev10016618m cation binding - like protein; c, Ciclev10025101m. sulfate transporter 2;1; d, Ciclev10024393m leucine-rich repeat transmembrane protein kinase