Summary Cross‐kingdom RNA interference (ckRNAi) is a mechanism of interspecies communication where small RNAs (sRNAs) are transported from one organism to another; these sRNAs silence target genes in trans by loading into host AGO proteins. In this work, we investigated the occurrence of ckRNAi in Arbuscular Mycorrhizal Symbiosis (AMS). We used an in silico prediction analysis to identify a sRNA (Rir2216) from the AM fungus Rhizophagus irregularis and its putative plant gene target, the Medicago truncatula MtWRKY69 transcription factor. Heterologous co‐expression assays in Nicotiana benthamiana, 5′ RACE reactions and AGO1‐immunoprecipitation assays from mycorrhizal roots were used to characterize the Rir2216–MtWRKY69 interaction. We further analyzed MtWRKY69 expression profile and the contribution of constitutive and conditional MtWRKY69 expression to AMS. We show that Rir2216 is loaded into an AGO1 silencing complex from the host plant M. truncatula, leading to cleavage of a host target transcript encoding for the MtWRKY69 transcription factor. MtWRKY69 is specifically downregulated in arbusculated cells in mycorrhizal roots and increased levels of MtWRKY69 expression led to a reduced AM colonization level. Our results indicate that MtWRKY69 silencing, mediated by a fungal sRNA, is relevant for AMS; we thus present the first experimental evidence of fungus to plant ckRNAi in AMS.

中文翻译：

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真菌 sRNA 沉默宿主植物转录因子以促进丛枝菌根共生


总结 跨界 RNA 干扰 （ckRNAi） 是一种种间通讯机制，其中小 RNA （sRNA） 从一个生物体转运到另一个生物体;这些 sRNA 通过加载到宿主 AGO 蛋白中来沉默反式靶基因。在这项工作中，我们研究了 ckRNAi 在丛枝菌根共生 （AMS） 中的发生。我们使用计算机预测分析来鉴定来自 AM 真菌 Rhizophagus irregularis 及其推定的植物基因靶标 Medicago truncatula MtWRKY69 转录因子的 sRNA （Rir2216）。使用本氏烟草中的异源共表达测定、5′ RACE 反应和菌根的 AGO1-免疫沉淀测定来表征 Rir2216-MtWRKY69 相互作用。我们进一步分析了 MtWRKY69 表达谱以及组成型和条件性 MtWRKY69 表达对 AMS 的贡献。我们表明 Rir2216 被加载到来自寄主植物 M. truncatula 的 AGO1 沉默复合物中，导致编码 MtWRKY69 转录因子的宿主靶转录物的切割。MtWRKY69 在菌根的丛枝细胞中特异性下调，MtWRKY69 表达水平增加导致 AM 定植水平降低。我们的结果表明，由真菌 sRNA 介导的 MtWRKY69 沉默与 AMS 相关;因此，我们提出了真菌在 AMS 中种植 ckRNAi 的第一个实验证据。