Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

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.

Tobacco mosaic virus and the virescence of biotechnology

“…High levels of phytoene also accumulated in photobleached, transfected leaves of N. benthamiana by silencing the expression of the downstream enzyme phytoene desaturase using fragments of a gene derived from tomato and cloned in either the sense or antisense orientation (Kumagai et al 1995). Therefore, the virus-induced gene silencing originally observed to target viral-derived transgenes (Lindbo et al 1993) can be widely and conveniently applied in functional genomics studies.…”

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…It has been reported that VIGS of PDS resulted in a photobleached phenotype (Kumagai et al, 1995;Liu et al, 2002a). Two-leaf-stage barley plants were inoculated with saps of N. benthamiana leaves agroinfiltrated with FoMV-HvPDS200 or empty vector pFoMV-sg.…”

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

“…The cDNA fragments (0.5 kb) corresponding N. benthamiana (Nb SAMT1) and Pantoea agglomerans phytoene synthase (CrtB; P22872), used as a control, were amplified by PCR and cloned into the viral vector TTO (Kumagai et al, 1995). For TTO-Nb SAMT1, the forward primer 59-GCCTCGAGTTGAAGATGCTTCCTGAAAATCTTAGTGC-39 and the reverse primer 5-TCCCTAGGCCAAAGCACTCTTGGACCGATGCCCTT-39 containing XhoI and AvrII restriction sites, respectively, were used.…”