Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena, Germany.
PLoS One. 2012;7(2):e31347. doi: 10.1371/journal.pone.0031347. Epub 2012 Feb 1.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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.
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.
通过喂食稳定转化表达所选中肠表达基因双链 RNA (dsRNA) 的宿主植物，昆虫草食动物可以实现 RNAi。然而，稳定转化植物的发展是一个缓慢而费力的过程，在这里我们开发了一种快速、可靠和瞬时的方法。我们使用病毒载体在宿主植物 Nicotiana attenuata 中产生 dsRNA，以瞬时沉默植物鳞翅目专食性草食动物 Manduca sexta 中肠基因。为了比较较长、未切割 dsRNA 对昆虫基因沉默的效果，我们在稳定转化表达靶向昆虫基因 dsRNA 的植物中沉默了 N. attenuata 的 dicer 基因 (NaDCL1-4) 的所有组合。
方法/主要发现：生成了稳定转化的 N. attenuata 植物，其携带 MsCYP6B46 的 312 bp 片段以反向重复取向（ir-CYP6B46），以产生 CYP6B46 dsRNA。食用这些植物后，M. sexta 幼虫中肠的 CYP6B46 转录本显著减少。相同的 312 bp cDNA 以反义取向克隆到 TRV 载体中，并农杆菌浸润到 N. attenuata 植物中。当幼虫食用这些植物时，观察到 CYP6B46 转录本的相似减少，而没有减少最密切相关的 MsCYP6B45 的转录本。我们使用这种瞬时方法快速沉默另外两个中肠表达的 MsCYPs 的表达。当幼虫食用瞬时沉默两个 NaDCLs 组合（DCL1/3/4 和 DCL2/3/4）并含有更高浓度的未切割 CYP6B46 dsRNA 的 ir-CYP6B46 植物时，CYP6B46 转录本在中肠中进一步减少。
宿主植物中 CYP6B46 dsRNA 的稳定和瞬时表达都为 M. sexta 幼虫中该基因的沉默提供了一种特异而强大的手段，但瞬时系统更适合高通量分析。在 ir-CYP6B46 植物中瞬时沉默 NaDCLs 增加了 MsCYP6B46 的沉默，表明昆虫的 RNAi 机制对摄入的 dsRNA 长度更长更有效。
