Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein
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 overexpres...
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| Veröffentlicht in: | PloS one 2012-12, Vol.7 (12), p.e52717 |
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| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0052717 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0052717</identifier><identifier>PMID: 23300750</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Agriculture ; Analysis ; Arabidopsis ; Assaying ; Begomovirus - genetics ; Biology ; Desaturation ; Elongation ; Engineering ; Enzymes ; Fatty Acid Desaturases - genetics ; Fatty Acid Desaturases - metabolism ; Fatty acids ; Gene expression ; Gene Expression Regulation, Plant ; Gene Knockdown Techniques ; Genes, Viral ; Genetic Engineering ; Genome, Plant ; Genomes ; Genomics ; High-Throughput Nucleotide Sequencing ; Industrial research ; Inverted Repeat Sequences ; Leaves ; Lipid metabolism ; Lipid Metabolism - genetics ; Lipids ; Metabolic engineering ; Metabolism ; Metabolites ; Nicotiana - enzymology ; Nicotiana - genetics ; Nicotiana benthamiana ; Physiology ; Plant diseases ; Plant Leaves - enzymology ; Plant Leaves - genetics ; Plant Oils - metabolism ; Plant Proteins - genetics ; Plant Proteins - metabolism ; Plants, Genetically Modified - enzymology ; Plants, Genetically Modified - genetics ; Polymerase chain reaction ; Proteins ; Ribonucleic acid ; RNA ; RNA, Small Interfering - genetics ; RNA-mediated interference ; Sequence Analysis, DNA ; siRNA ; Substrates ; Tomatoes ; Transgenes ; Viruses ; Yellow leaf</subject><ispartof>PloS one, 2012-12, Vol.7 (12), p.e52717</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 Naim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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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. 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23300750</pmid><doi>10.1371/journal.pone.0052717</doi><tpages>e52717</tpages><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1327225064 |
| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Agriculture Analysis Arabidopsis Assaying Begomovirus - genetics Biology Desaturation Elongation Engineering Enzymes Fatty Acid Desaturases - genetics Fatty Acid Desaturases - metabolism Fatty acids Gene expression Gene Expression Regulation, Plant Gene Knockdown Techniques Genes, Viral Genetic Engineering Genome, Plant Genomes Genomics High-Throughput Nucleotide Sequencing Industrial research Inverted Repeat Sequences Leaves Lipid metabolism Lipid Metabolism - genetics Lipids Metabolic engineering Metabolism Metabolites Nicotiana - enzymology Nicotiana - genetics Nicotiana benthamiana Physiology Plant diseases Plant Leaves - enzymology Plant Leaves - genetics Plant Oils - metabolism Plant Proteins - genetics Plant Proteins - metabolism Plants, Genetically Modified - enzymology Plants, Genetically Modified - genetics Polymerase chain reaction Proteins Ribonucleic acid RNA RNA, Small Interfering - genetics RNA-mediated interference Sequence Analysis, DNA siRNA Substrates Tomatoes Transgenes Viruses Yellow leaf |
| title | Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-18T23%3A20%3A06IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Advanced%20engineering%20of%20lipid%20metabolism%20in%20Nicotiana%20benthamiana%20using%20a%20draft%20genome%20and%20the%20V2%20viral%20silencing-suppressor%20protein&rft.jtitle=PloS%20one&rft.au=Naim,%20Fatima&rft.date=2012-12-26&rft.volume=7&rft.issue=12&rft.spage=e52717&rft.pages=e52717-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0052717&rft_dat=%3Cgale_plos_%3EA477041380%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1327225064&rft_id=info:pmid/23300750&rft_galeid=A477041380&rft_doaj_id=oai_doaj_org_article_b2be3330261a4f06a241d54c059bd915&rfr_iscdi=true |