Transcriptional changes in Nicotiana benthamiana induced by tobamoviral transfection

This research has been dedicated to the development of a system to study the utility of plant viral vector technology for metabolic engineering and functional genomic applications. Potato cDNA microarrays developed by The Institute for Genomic Research (TIGR) were employed to determine gene expression changes in transfected Nicotiana benthamiana plants. Several challenges were addressed in developing the system, such as selection of appropriate controls; subtracting out effects of virus infection on plant transcription; and determining the effectiveness of using heterologous microarrays for solanaceous species.; Regulation of carotenoid biosynthesis was investigated by inoculating plants with TTU5I/CTP-CrtB-RZ carrying a phytoene synthase cDNA derived from Erwinia herbicola. Microarray analysis showed that the expression of genes encoding enzymes in leaf carotenogenesis was upregulated and that transcripts both upstream (isopentenyl diphosphate isomerase) and downstream (beta-carotene hydroxylase) of the targeted enzyme accumulated at ten days post-inoculation (dpi). Quantitative real-time PCR (QRT-PCR) data validated an elevation of endogenous phytoene synthase and phytoene desaturase (pds) mRNAs. Plants transfected with a TTO1/PDS- viral vector carrying a partial pds antisense construct, showed a 5-fold decrease in transcript levels of a putative 9-cisepoxycarotenoid dioxygenase (NCED) and a 78-fold decrease in pds mRNA compared to controls. These data demonstrate that tobamoviral vectors are valuable tools in the metabolic engineering of plant pathways. Accumulation of colorless phytoene in both transfections does not appear to play a role in pathway regulation.; In a reverse genetics approach, a time course analysis was conducted for N. benthamiana transfected with 740AT#120 viral vector carrying an antisense Arabidopsis ADP-ribosylation factor-1 (ARF-1). Cytoplasmic inhibition of gene expression produced a progressively stunted phenotype. A Western analysis showed that ARF protein levels gradually decline and that all members of this multigene family are knocked down by 20 dpi. QRT-PCR shows endogenous ARF transcripts are 2.4x lower than control levels. A Welch ANOVA of microarray data identified sixteen genes for further characterization that have a potential role in the secretory or G-protein signaling pathways. Overall, these data suggest that combining viral vector and transcriptional profiling technologies is a useful strategy to rapidly filter gene expression data to assess gene function in plants.