Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

Naim, F., Nakasugi, K., Crowhurst, R.N., Hilario, E., Zwart, A.B., Hellens, R.P., Taylor, J.M., Waterhouse, P.M., & Wood, C.C. (2012) Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein. PLoS ONE, 7(12), e52717.

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The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18:1 to elongation reactions beginning with 18:1 as substrate. These V2-based leaf assays produced ~50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays. © 2012 Naim et al.

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ID Code: 65791
Item Type: Journal Article
Refereed: Yes
Keywords: fatty acid, lipid, short hairpin RNA, small interfering RNA, unclassified drug, V2 protein, virus protein, article, bioassay, controlled study, FAD2 gene, FAD2.1 gene, FAD2.2 gene, fatty acid desaturation, gene overexpression, gene silencing, genome, haploidy, lipid metabolism, metabolic engineering, multigene family, Nicotiana benthamiana, nonhuman, plant gene, plant leaf, plant virus, reverse transcription polymerase chain reaction, RNA interference, Tomato yellow leaf curl virus, transgene, transient leaf assay, Begomovirus, Fatty Acid Desaturases, Gene Expression Regulation, Plant, Gene Knockdown Techniques, Genes, Viral, Genetic Engineering, Genome, Plant, High-Throughput Nucleotide Sequencing, Inverted Repeat Sequences, Plant Leaves, Plant Oils, Plant Proteins, Plants, Genetically Modified, RNA, Small Interfering, Sequence Analysis, DNA, Tobacco
DOI: 10.1371/journal.pone.0052717
ISSN: 1932-6203
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000)
Divisions: Current > Schools > School of Earth, Environmental & Biological Sciences
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2012 Naim et al.
Copyright Statement: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Deposited On: 07 Jan 2014 23:17
Last Modified: 14 Mar 2016 22:17

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