Argonautes (AGOs) associate with noncoding RNAs to regulate gene expression during development and stress adaptation. Their role in plant immunity against hemibiotrophic fungal infection remains poorly understood. Here, we explore the function of AGOs in the interaction of wild tobacco (Nicotiana attenuata) with a naturally occurring hemibiotrophic pathogen, Fusarium brachygibbosum. Among all AGOs, only transcripts of AGO4 were elicited after fungal infection. The disease progressed more rapidly in AGO4-silenced (irAGO4) plants than in wild type, and small RNA (smRNA) profiling revealed that 24-nucleotide smRNA accumulation was severely abrogated in irAGO4 plants. Unique microRNAs (miRNAs: 130 conserved and 208 novel, including 11 canonical miRNA sequence variants known as "isomiRs") were identified in infected plants; silencing of AGO4 strongly changed miRNA accumulation dynamics. Time-course studies revealed that infection increased accumulation of abscisic acid, jasmonates, and salicylic acid in wild type; in irAGO4 plants, infection accumulated lower jasmonate levels and lower transcripts of jasmonic acid (JA) biosynthesis genes. Treating irAGO4 plants with JA, methyl jasmonate, or cis-(+)-12-oxo-phytodienoic acid restored wild-type levels of resistance. Silencing expression of RNA-directed RNA polymerases RdR1 and RdR2 (but not RdR3) and Dicer-like3 (DCL3, but not DCL2 or DCL4) increased susceptibility to F. brachygibbosum. The relevance of AGO4, RdR1, RdR2, and DCL3 in a natural setting was revealed when plants individually silenced in their expression (and their binary combinations) were planted in a diseased field plot in the Great Basin Desert of Utah. These plants were more susceptible to infection and accumulated lower JA levels than wild type. We infer that AGO4-dependent smRNAs play a central role in modulating JA biogenesis and signaling during hemibiotrophic fungal infections.

Argonautes (AGO) 与非编码 RNA 结合，在发育和应激适应过程中调节基因表达。它们在植物抵抗半生物营养真菌感染的免疫中的作用仍然知之甚少。在这里，我们探讨了 AGO 在野生烟草 ( Nicotiana attenuata ) 与天然存在的半生物营养病原体Fusarium brachygibbosum相互作用中的功能。在所有 AGO 中，真菌感染后仅诱导出AGO4的转录本。与野生型相比，AGO4 沉默 (irAGO4) 植物中的疾病进展更快，小 RNA (smRNA) 分析显示 irAGO4 植物中 24 核苷酸 smRNA 的积累被严重消除。在受感染的植物中鉴定出独特的 microRNA（miRNA：130 个保守的 miRNA 和 208 个新的 miRNA，包括 11 个被称为“isomiR”的规范 miRNA 序列变体）；AGO4 的沉默强烈改变了 miRNA 积累动态。时程研究表明，感染增加了野生型中脱落酸、茉莉酸和水杨酸的积累；在 irAGO4 植物中，感染积累了较低的茉莉酸水平和较低的茉莉酸 (JA) 生物合成基因转录本。用 JA、茉莉酸甲酯或顺式-(+)-12-氧代-植物二烯酸处理 irAGO4 植物可恢复野生型水平的抗性。沉默 RNA 指导的 RNA 聚合酶RdR1和RdR2（但不是RdR3）和Dicer - like3（DCL3，但不是DCL2或DCL4）的表达会增加对F的敏感性。短臂藻。当将表达单独沉默的植物（及其二元组合）种植在犹他州大盆地沙漠的患病田地中时，揭示了 AGO4 、 RdR1 、 RdR2 和DCL3在自然环境中的相关性。这些植物比野生型更容易受到感染，并且积累的 JA 水平较低。我们推断，AGO4 依赖性 smRNA 在半生物营养型真菌感染期间调节 JA 生物发生和信号传导中发挥着核心作用。