National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, China.
Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China.
Mol Plant Pathol. 2023 Aug;24(8):961-972. doi: 10.1111/mpp.13344. Epub 2023 Apr 28.
RNA silencing, a core part of plants' antiviral defence, requires the ARGONAUTE, DICER-like, and RNA-dependent RNA polymerase proteins. However, how these proteins contribute to watermelon's RNA interference (RNAi) pathway response to cucumber green mottle mosaic virus (CGMMV) has not been characterized. Here, we identify seven ClAGO, four ClDCL, and 11 ClRDR genes in watermelon and analyse their expression profiles when infected with CGMMV. ClAGO1 and ClAGO5 expression levels were highly induced by CGMMV infection. The results of ClAGO1 and ClAGO5 overexpression and silencing experiments suggest that these genes play central roles in watermelon's antiviral defence. Furthermore, co-immunoprecipitation and bimolecular fluorescence complementation experiments showed that ClAGO1 interacts with ClAGO5 in vivo, suggesting that ClAGO1 and ClAGO5 co-regulate watermelon defence against CGMMV infection. We also identified the ethylene response factor (ERF) binding site in the promoters of the ClAGO1 and ClAGO5 genes, and ethylene (ETH) treatment significantly increased ClAGO5 expression. Two ERF genes (Cla97C08G147180 and Cla97C06G122830) closely related to ClAGO5 expression were identified using co-expression analysis. Subcellular localization revealed that two ERFs and ClAGO5 predominantly localize at the nucleus, suggesting that enhancement of resistance to CGMMV by ETH is probably achieved through ClAGO5 but not ClAGO1. Our findings reveal aspects of the mechanisms underlying RNA silencing in watermelon against CGMMV.
RNA 沉默是植物抗病毒防御的核心部分，需要 ARGONAUTE、DICER 样和 RNA 依赖性 RNA 聚合酶蛋白。然而，这些蛋白质如何有助于西瓜的 RNA 干扰（RNAi）途径对黄瓜绿斑驳花叶病毒（CGMMV）的反应尚未得到描述。在这里，我们在西瓜中鉴定了 7 个 ClAGO、4 个 ClDCL 和 11 个 ClRDR 基因，并分析了它们在感染 CGMMV 时的表达谱。ClAGO1 和 ClAGO5 的表达水平受到 CGMMV 感染的高度诱导。ClAGO1 和 ClAGO5 过表达和沉默实验的结果表明，这些基因在西瓜的抗病毒防御中发挥着核心作用。此外，共免疫沉淀和双分子荧光互补实验表明，ClAGO1 在体内与 ClAGO5 相互作用，表明 ClAGO1 和 ClAGO5 共同调节西瓜对 CGMMV 感染的防御。我们还鉴定了 ClAGO1 和 ClAGO5 基因启动子中的乙烯响应因子（ERF）结合位点，并且 ETH 处理显著增加了 ClAGO5 的表达。通过共表达分析鉴定了与 ClAGO5 表达密切相关的两个 ERF 基因（Cla97C08G147180 和 Cla97C06G122830）。亚细胞定位表明，两个 ERFs 和 ClAGO5 主要定位于细胞核，表明 ETH 通过 ClAGO5 而不是 ClAGO1 增强对 CGMMV 的抗性。我们的发现揭示了西瓜对 CGMMV 进行 RNA 沉默的机制的一些方面。
