title: Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein.
abstract: 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.
authors.affiliation: Plant Industry, The Commonwealth Scientific and Industrial Research Organisation, Canberra, Australian Capital Territory, Australia.
authors.forename: Fatima
authors.initials: F
authors.lastname: Naim
authors.forename: Kenlee
authors.initials: K
authors.lastname: Nakasugi
authors.forename: Ross N
authors.initials: RN
authors.lastname: Crowhurst
authors.forename: Elena
authors.initials: E
authors.lastname: Hilario
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Zwart
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hellens
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Taylor
authors.forename: Peter M
authors.initials: PM
authors.lastname: Waterhouse
authors.forename: Craig C
authors.initials: CC
authors.lastname: Wood
mesh_terms: D053839:Begomovirus / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D055785:Gene Knockdown Techniques; D005814:Genes, Viral; D005818:Genetic Engineering; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing; D055029:Inverted Repeat Sequences; D050356:Lipid Metabolism / Q000235:genetics*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics*; D010938:Plant Oils / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
chemical_list: D010938:Plant Oils; D010940:Plant Proteins; D034741:RNA, Small Interfering; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase