Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.

<h4>Background</h4>RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we develop...

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Autores principales:	Pavan Kumar, Sagar Subhash Pandit, Ian T Baldwin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/98a4835427194698bf34ad4f12d6b94c
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spelling	oai:doaj.org-article:98a4835427194698bf34ad4f12d6b94c2021-11-18T07:29:02ZTobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.1932-620310.1371/journal.pone.0031347https://doaj.org/article/98a4835427194698bf34ad4f12d6b94c2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22312445/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.<h4>Methodology/principal findings</h4>Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.<h4>Conclusions</h4>Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.Pavan KumarSagar Subhash PanditIan T BaldwinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e31347 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pavan Kumar Sagar Subhash Pandit Ian T Baldwin Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
description	<h4>Background</h4>RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.<h4>Methodology/principal findings</h4>Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.<h4>Conclusions</h4>Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.
format	article
author	Pavan Kumar Sagar Subhash Pandit Ian T Baldwin
author_facet	Pavan Kumar Sagar Subhash Pandit Ian T Baldwin
author_sort	Pavan Kumar
title	Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
title_short	Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
title_full	Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
title_fullStr	Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
title_full_unstemmed	Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
title_sort	tobacco rattle virus vector: a rapid and transient means of silencing manduca sexta genes by plant mediated rna interference.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/98a4835427194698bf34ad4f12d6b94c
work_keys_str_mv	AT pavankumar tobaccorattlevirusvectorarapidandtransientmeansofsilencingmanducasextagenesbyplantmediatedrnainterference AT sagarsubhashpandit tobaccorattlevirusvectorarapidandtransientmeansofsilencingmanducasextagenesbyplantmediatedrnainterference AT iantbaldwin tobaccorattlevirusvectorarapidandtransientmeansofsilencingmanducasextagenesbyplantmediatedrnainterference
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Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.

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