RNA干扰(RNAi)尚未在大流行两栖动物病原体中进行测试,Batrachochytriumdendrobatidis,但是开发这种技术可能有助于阐明毒力机制,确定治疗目标,并可能为乳糜菌病提供新的抗真菌治疗选择。为了操纵和破译基因功能,合理设计的小干扰RNA(siRNA)可以启动同源信使RNA(mRNA)的破坏,导致靶基因表达的“敲低”。这里,我们研究了siRNA是否可用于通过RNAi操纵树枝状芽孢杆菌的基因表达,使用不同的siRNA策略靶向参与谷胱甘肽和鸟氨酸合成的基因.为了确定mRNA敲低的程度和持续时间,在递送靶向谷氨酸-半胱氨酸连接酶的siRNA后24-48小时监测靶mRNA水平,在36小时时,靶转录物最多减少约56%。靶向谷氨酸-半胱氨酸连接酶的第二siRNA设计在该时间点也导致约53%的敲减。针对不同基因靶标的siRNA,鸟氨酸脱羧酶,目标转录物减少了17%。虽然没有观察到表型效应,这些结果表明RNAi在树枝状芽孢杆菌中是可能的,基因表达可以在这种病原体中被操纵。我们概述了进一步优化步骤的想法,以提高敲除效率,从而更好地利用RNAi技术来控制树枝状芽孢杆菌。
RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the \"knockdown\" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.