Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

TLDR

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. A tobamovirus subgenomic promoter drives expression of a phytoene synthase cDNA and a partial phytoene desaturase cDNA, and infected Nicotiana benthamiana plants were examined 1–2 weeks post‑inoculation for phytoene accumulation. Phytoene synthase expression yielded bright orange leaves with elevated phytoene, whereas antisense PDS transcripts produced white leaves with high phytoene, confirming cytoplasmic inhibition of endogenous gene expression.

Abstract

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.