Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
J Insect Physiol. 2013 Jun;59(6):646-54. doi: 10.1016/j.jinsphys.2013.03.013. Epub 2013 Apr 17.
Injection of dsRNA is widely applied to silence endogenous genes and study gene function in insects. However, it is not yet clear to what extent it can also exert non-specific effects, for instance by interference with the innate immune response. In this study, we report on the transcriptional response of BmToll9-1 to lipopolysaccharide (LPS) and dsRNA in the silkmoth, Bombyx mori. BmToll9-1 encodes a Toll receptor highly expressed in midgut tissue and that shows limited similarity to the mammalian TLR3 endolysosome receptor for dsRNA; while Dcr2 and Ago2 encode two key components of the RNAi machinery. An expression pattern study of all 14 Toll receptors in B. mori showed that BmToll9-1 was expressed in different larval and pupal tissues with the highest expression level detected in the midgut, indicating a possible function in immunity against pathogens taken up by the food. In order to investigate the response of BmToll9-1, different ways to deliver dsRNA, specific for GFP (dsGFP), and LPS were applied in Bombyx 5th instar larvae. The feeding experiments suggested that dsGFP did not suppress the expression of BmToll9-1 significantly, while LPS could suppress the expression of BmToll9-1 after 3h of feeding. On the other hand, the injection experiments showed that dsGFP, as well as LPS, could significantly inhibit the expression of BmToll9-1 in 3h. Bacteria that constantly expressed dsGFP could also down-regulate the expression of BmToll9-1 to a greater extent than bacteria that do not express dsGFP. The failure of dsGFP by feeding to affect the expression of BmToll9-1 was correlated with the rapid degradation of dsGFP by dsRNase in the midgut juice. Expression of the RNAi machinery genes Dcr2 and Ago2, as well as dsRNase, was also affected by injection of dsRNA and not by feeding, but in these cases an induction was observed instead of a down-regulation. Because LPS is a well-known pathogen-associated molecular pattern (PAMP), it suggested that the decrease in BmToll9-1 expression is a consequence of the activation of the innate immune response by LPS. The similar response of BmToll9-1 between the two triggers, LPS and dsRNA, suggests that dsRNA can also act as a PAMP in the midgut of Bombyx. Furthermore, induction of the genes Dcr2, Ago2 and dsRNase may also constitute a defense mechanism against invading dsRNA.
双链 RNA 的注射被广泛应用于沉默内源性基因并研究昆虫中的基因功能。然而，目前尚不清楚它在多大程度上也会产生非特异性效应，例如通过干扰先天免疫反应。在这项研究中，我们报告了家蚕中 BmToll9-1 对脂多糖（LPS）和双链 RNA 的转录反应。BmToll9-1 编码一种 Toll 受体，在中肠组织中高度表达，与哺乳动物 TLR3 内体受体对双链 RNA 的相似性有限；而 Dcr2 和 Ago2 编码 RNAi 机制的两个关键成分。对家蚕 14 种 Toll 受体的表达模式研究表明，BmToll9-1 在不同的幼虫和蛹组织中表达，在中肠中检测到最高的表达水平，表明其可能在针对食物中摄取的病原体的免疫中发挥作用。为了研究 BmToll9-1 的反应，我们以不同的方式向 5 龄家蚕幼虫体内注射靶向 GFP（dsGFP）和 LPS 的双链 RNA。喂食实验表明，dsGFP 并没有显著抑制 BmToll9-1 的表达，而 LPS 在喂食 3 小时后可以抑制 BmToll9-1 的表达。另一方面，注射实验表明，dsGFP 和 LPS 都可以在 3 小时内显著抑制 BmToll9-1 的表达。持续表达 dsGFP 的细菌也可以比不表达 dsGFP 的细菌更大程度地下调 BmToll9-1 的表达。dsGFP 通过喂食影响 BmToll9-1 表达的失败与 dsGFP 在中肠液中被 dsRNase 快速降解有关。RNAi 机制基因 Dcr2 和 Ago2 的表达以及 dsRNase 的表达也受到双链 RNA 注射的影响，而不是通过喂食，但是在这些情况下观察到的是诱导而不是下调。由于 LPS 是一种众所周知的病原体相关分子模式（PAMP），这表明 LPS 激活先天免疫反应会导致 BmToll9-1 表达的减少。LPS 和 dsRNA 这两种触发因素对 BmToll9-1 的相似反应表明，dsRNA 也可以作为家蚕中肠的 PAMP。此外，Dcr2、Ago2 和 dsRNase 基因的诱导也可能构成对抗入侵 dsRNA 的防御机制。
